Genetically modified cells and methods for expressing recombinant heparanase and methods of purifying same

ABSTRACT

A preparation containing an enzymatically inactive form of heparanase, said enzymatically inactive form of heparanase being cleavable into an enzimatically active form of heparanase.

This is a continuation of U.S. patent application Ser. No. 09/487,716,filed Jan. 19, 2000, which is a continuation of U.S. patent applicationSer. No. 09/260,038, filed Mar. 2, 1999, now U.S. Pat. No. 6,348,344,issued Feb. 19, 2002, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/071,618 filed May 1, 1998 now abandoned, whichis a continuation-in-part of U.S. patent application Ser. No.09/071,739, filed May 1, 1998, now U.S. Pat. No. 6,177,545, issued Jan.23, 2001 which is a continuation-in-part of U.S. patent application Ser.No. 08/922,170, filed Sep. 2, 1997 now U.S. Pat. No. 5,968,822, issuedOct. 19, 1999.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to genetically modified cellsoverexpressing recombinant heparanase, to methods of overexpressingrecombinant heparanase in cellular systems and to methods of purifyingrecombinant heparanase. The invention further relates to nucleic acidconstructs for directing the expression of modified heparanase speciesto which a protease recognition and cleavage sequence has beenintroduced, to the modified heparanase species expressed therefrom andto their proteolytic products. The invention further relates to in vivomethods of inhibiting heparanase activity.

The extracellular matrix (ECM) acts both as a structural scaffold and asan informational medium. Its dynamic status is determined by cells thatsecrete both its constituent molecules and enzymes that catalyze thedegradation of these molecules. A stasis between ECM degrading enzymesand their inhibitors maintains the integrity of the matrix. Whilecontrolled ECM remodeling is fundamental to normal processes,uncontrolled disruption underlies diverse pathological conditions.

Among the integral constituents of basement membrane and ECM are celladhesion molecules such as laminin and fibronectin, structuralcomponents like collagen and ellastin, and proteoglycans includingsydecan, serglican, proteoglycan I and II versican (1-2).

BRIEF OVERVIEW ON RECOMBINANT GENE EXPRESSION

For biochemical characterization of a protein and pharmaceuticalapplications, it is often necessary to overproduce and purify largequantities of the protein. A major consideration when setting up aproduction scheme for a recombinant protein is whether the productshould be expressed intracellularly or if a secretion system can be usedto direct the protein to the growth medium. The inherent properties ofthe protein and the intended applications dictate the expression systemof choice. Another consideration when attempting the production ofrecombinant eukaryotic proteins are the folding and post translationalmodification processes associated with their natural expression.

Preferably, production is carried out in a cellular system that supportsappropriate transcription, translation, and post-translationmodification of the protein of interest. Thus, cultured mammalian cellsare widely used in applied biotechnology as well as in differentdisciplines of basic sciences of cellular and molecular biology forproducing recombinant proteins of mammalian origin.

One of the most widely used cells for recombinant protein expression,particularly for biotechnological applications, is the Chinese hamsterovary cell line (CHO). Alternatively, baby hamster kidney cells (BHK21),Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells,Ehrlich's ascites cells, Sk-Hep1 cells, MDCK₁ cells, MDBK₁ cells, Verocells, Cos cells, CV-1 cells, NIH3T3 cells, L929 cells and BLG cells(mouse melanoma) have also been shown to consecutively express largequantities of recombinant proteins.

These cells are easily transfected with foreign DNA, that can integrateinto the host genome to create stable cell lines, with new acquiredcharacteristics (i.e. expression of recombinant proteins). These newcell lines originate from a single cell that has undergone foreign DNAincorporation and are therefore referred to as “cellular clones”.

Since integration of foreign DNA in host cell genome is relativelyinefficient, the isolation of cellular clones requires a selectionsystem that discriminates between the stably transformed and the primarycells.

Dihydrofolate reductase deficiency in CHO cells (CHO dhfr-cell line)offers a particularly convenient selection system for cellular clones.Transfection of the dhfr gene along with the gene of interest, resultsin the survival of clones in a growth medium containing methotrexate(MTX). The higher the number of foreign dhfr gene copies in the cellularclone, the higher the MTX concentration the cells can survive. It hasbeen demonstrated that integration events of foreign DNA into host cellgenome often maintain all the components of the transfected DNA. e.g.,the selection marker as well as the gene of interest (67).

In contrast to mammalian expression systems, that inherently expresslimited quantities of recombinant proteins, other expression systems,such as bacteria, yeast, and virus infected insect cells are widelyused.

Using such cellular gene expression systems, large amounts of eitheractive or non-active protein can be obtained and used for biochemicalanalysis of protein properties, structure function relationship, kineticstudies, identification of, screening for, or production of specificinhibitors, production of poly- and monoclonal antibodies recognizingthe protein, pharmaceutical applications and the like.

Bacteria are the most powerful tool for the production of recombinantproteins. A recombinant protein that is overproduced in a bacterialsystem might constitute up to 30% of the total protein content of thecells. The recombinant protein accumulates in inclusion bodies where itis relatively pure (comprises up to 50% of the protein content of thebodies) and protected from protease degradation.

Inclusion bodies enable the accumulation of up to 0.2 grams of proteinper liter fermentation culture.

Using specific expression vectors, bacteria can also be directed toproduce and secrete proteins into the periplasm and therefrom into thegrowth medium. Although the reported production quantities are not ashigh as in inclusion bodies, purification of the expressed protein maybe simpler (68).

These advantages and the relative simple growth conditions required forbacteria to thrive, made bacteria a powerful and widely used cellularexpression system for the production of recombinant proteins of interest(e.g., human -interferon, human β-interferon, GM-CSF, G-CSF, humanLNF-γ, IL-2, IL-3, IL-6, TNF, human insulin, human growth hormone,etc.).

Furthermore, non-active bacterialy produced recombinant proteins due toinappropriate folding and disulfide bonding may be reduced and/ordenatured and thereafter deoxidized and/or refolded to acquire thecatalytically active conformation.

However, when glycosylation of the protein is essential for its activityor uses, eukaryotic expression systems are required.

Yeasts are eukaryotic microorganisms which are widely used forcommercial production of recombinant proteins. Examples include theproduction of insulin, human GM-CSF and hepatitis B antigens (forvaccination) by the yeast Saccharomyces cerevisiae. The relativelysimple growth conditions and the fact that yeasts are eukaryotes makethe yeast gene expression system highly suitable for the production ofrecombinant proteins, primarily those with pharmaceutical relevance.

In recent years methylotrophic yeasts (e.g., Pichia pastoris, Hansenulapolymorpha) became widely used, thus replacing in many cases the moretraditionally used yeast Saccharomyces cerevisiae.

Methylotrophic yeasts can grow to a high cellular density, and expressand if appropriately, secrete, high levels of recombinant proteins.Quantities of the secreted, correctly-folded recombinant protein canaccumulate up to several grams per liter culture. These advantages makePichia pastoris suitable for an efficient production of recombinantproteins (69).

One aspect of the present invention thus concerns the expression ofrecombinant heparanase in cellular systems.

Heparan Sulfate Proteoglycans (HSPGs)

HSPGs are ubiquitous macromolecules associated with the cell surface andextracellular matrix (ECM) of a wide range of cells of vertebrate andinvertebrate tissues (3-7). The basic HSPG structure consists of aprotein core to which several linear heparan sulfate chains arecovalently attached. The polysaccharide chains are typically composed ofrepeating hexuronic and D-glucosamine disaccharide units that aresubstituted to a varying extent with N- and O-linked sulfate moietiesand N-linked acetyl groups (3-7). Studies on the involvement of ECMmolecules in cell attachment, growth and differentiation revealed acentral role of HSPGs in embryonic morphogenesis, angiogenesis,metastasis, neurite outgrowth and tissue repair (3-7). The heparansulfate (HS) chains, which are unique in their ability to bind amultitude of proteins, ensure that a wide variety of effector moleculescling to the cell surface (6-8). HSPGs are also prominent components ofblood vessels (5). In large vessels they are concentrated mostly in theintima and inner media, whereas in capillaries they are found mainly inthe subendothelial basement membrane where they support proliferatingand migrating endothelial cells and stabilize the structure of thecapillary wall. The ability of HSPGs to interact with ECM macromoleculessuch as collagen, laminin and fibronectin, and with different attachmentsites on plasma membranes suggests a key role for this proteoglycan inthe self-assembly and insolubility of ECM components, as well as in celladhesion and locomotion. Cleavage of HS may therefore result indisassembly of the subendothelial ECM and hence may play a decisive rolein extravasation of normal and malignant blood-borne cells (9-11). HScatabolism is observed in inflammation, wound repair, diabetes, andcancer metastasis, suggesting that enzymes which degrade HS playimportant roles in pathologic processes.

Heparanase

Heparanase is a glycosylated enzyme that is involved in the catabolismof certain glycosaminoglycans. It is an endo-β-glucuronidase thatcleaves heparan sulfate at specific intrachain sites (12-15).Interaction of T and B lymphocytes, platelets, granulocytes, macrophagesand mast cells with the subendothelial extracellular matrix (ECM) isassociated with degradation of heparan sulfate by heparanase activity(16). Connective tissue activating peptide III (CTAP), an -chemokine,was found to have heparanase-like activity. Placenta heparanase acts asan adhesion molecule or as a degradative enzyme depending on the pH ofthe microenvironvent (17).

Heparanase is released from intracellular compartments (e.g., lysosomes,specific granules) in response to various activation signals (e.g.,thrombin, calcium ionophores, immune complexes, antigens and mitogens),suggesting its regulated involvement in inflammation and cellularimmunity responses (16).

It was also demonstrated that heparanase can be readily released fromhuman neutrophils by 60 minutes incubation at 4° C. in the absence ofadded stimuli (18).

Gelatinase, another ECM degrading enzyme which is found in tertiarygranules of human neutrophils with heparanase, is secreted from theneutrophils in response to phorbol 12-myristate 13-acetate (PMA)treatment (19-20).

In contrast, various tumor cells appear to express and secreteheparanase in a constitutive manner in correlation with their metastaticpotential (21).

Degradation of heparan sulfate by heparanase results in the release ofheparin-binding growth factors, enzymes and plasma proteins that aresequestered by heparan sulfate in basement membranes, extracellularmatrices and cell surfaces (22-23).

Purification of Natural Heparanase

Heparanase activity has been described in a number of cell typesincluding cultured skin fibroblasts, human neutrophils, activated ratT-lymphocytes, normal and neoplastic murine B-lymphocytes, humanmonocytes and human umbilical vein endothelial cells, SK hepatoma cells,human placenta and human platelets.

A procedure for purification of natural heparanase was reported for SKhepatoma cells and human placenta (U.S. Pat. No. 5,362,641) and forhuman platelets derived enzymes (62). Purification was performed by acombination of ion exchange and various affinity columns including Con-ASepharose, Blue A-agarose, Zn⁺⁺-chelating agarose and Heparin-Sepharose.Evidently, the amounts of active heparanase recovered by these methodsis low.

Cloning and Expression of the Heparanase Gene

A purified fraction of heparanase isolated from human hepatoma cells wassubjected to tryptic digestion. Peptides were separated by high pressureliquid chromatography (HPLC) and micro sequenced. The sequence of one ofthe peptides was used to screen data bases for homology to thecorresponding back translated DNA sequence. This procedure led to theidentification of a clone containing an insert of 1020 base pairs (bp)which included an open reading frame of 963 bp followed by 27 bp of 3′untranslated region and a poly A tail. The new gene was designated hpa.Cloning of the missing 5′ end of hpa was performed by PCR amplificationof DNA from placenta cDNA composite. The joined hpa cDNA (also referredto as phpa) fragment contained an open reading frame which encodes apolypeptide of 543 amino acids with a calculated molecular weight of61,192 daltons. Cloning an extended 5′ sequence was enabled from thehuman SK-hep1 cell line by PCR amplification using the Marathon RACEsystem. The 5′ extended sequence of the SK-hep1 hpa cDNA was assembledwith the sequence of the hpa cDNA isolated from human placenta. Theassembled sequence contained an open reading frame which encodes apolypeptide of 592 amino acids with a calculated molecular weight of66,407 daltons. The cloning procedures are described in length in U.S.patent application Ser. Nos. 08/922,170, 09/109,386, and 09/071,618,entitled POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASEACTIVITY AND EXPRESSION OF SAME IN GENETICALLY MODIFIED CELLS, which isa continuation-in-part of PCT/US98/17954, filed Aug. 31, 1998, all ofwhich are incorporated herein by reference.

The ability of the hpa gene product to catalyze degradation of heparansulfate (HS) in vitro was examined by expressing the entire open readingframe of hpa in High five and Sf21 insect cells, and the mammalian human293 embryonic kidney cell line expression systems. Extracts of infectedcells were assayed for heparanase catalytic activity. For this purpose,cell lysates were incubated with sulfate labeled, ECM-derived HSPG (peakI), followed by gel filtration analysis (Sepharose 6B) of the reactionmixture. While the substrate alone consisted of high molecular weightmaterial, incubation of the HSPG substrate with lysates of cellsinfected with hpa containing virus resulted in a complete conversion ofthe high molecular weight substrate into low molecular weight labeledheparan sulfate degradation fragments (see, for example, U.S. patentapplication Ser. No. 09/071,618, which is incorporated herein byreference.

In subsequent experiments, the labeled HSPG substrate was incubated withthe culture medium of infected High Five and Sf21 cells. Heparanasecatalytic activity, reflected by the conversion of the high molecularweight HSPG substrate into low molecular weight HS degradationfragments, was found in the culture medium of cells infected with thepFhpa virus, but not the control pF1 virus.

Altogether, these results indicate that the heparanase enzyme isexpressed in an active form by cells infected with Baculovirus ormammalian expression vectors containing the newly identified human hpagene.

In other experiments, it was demonstrated that the heparanase enzymeexpressed by cells infected with the pFhpa virus is capable of degradingHS complexed to other macromolecular constituents (e.g., fibronectin,laminin, collagen) present in a naturally produced intact ECM (see U.S.patent application Ser. No. 09/109,386, which is incorporated herein byreference), in a manner similar to that reported for highly metastatictumor cells or activated cells of the immune system (7, 8)

Involvement of Heparanase in Tumor Cell Invasion and Metastasis

Circulating tumor cells arrested in the capillary beds often attach ator near the intercellular junctions between adjacent endothelial cells.Such attachment of the metastatic cells is followed by rupture of thejunctions, retraction of the endothelial cell borders and migrationthrough the breach in the endothelium toward the exposed underlying basemembrane (BM) (24). Once located between endothelial cells and the BM,the invading cells must degrade the subendothelial glycoproteins andproteoglycans of the BM in order to migrate out of the vascularcompartment. Several cellular enzymes (e.g., collagenase IV, plasminogenactivator, cathepsin B, elastase, etc.) are thought to be involved indegradation of BM (25). Among these enzymes is heparanase that cleavesHS at specific intrachain sites (16,11). Expression of a HS degradingheparanase was found to correlate with the metastatic potential of mouselymphoma (26), fibrosarcoma and melanoma (21) cells. Moreover, elevatedlevels of heparanase were detected in sera from metastatic tumor bearinganimals and melanoma patients (21) and in tumor biopsies of cancerpatients (12).

The inhibitory effect of various non-anticoagulant species of heparin onheparanase was examined in view of their potential use in preventingextravasation of blood-borne cells. Treatment of experimental animalswith heparanase inhibitors markedly reduced (>90%) the incidence of lungmetastases induced by B16 melanoma, Lewis lung carcinoma and mammaryadenocarcinoma cells (12, 13, 28). Heparin fractions with high and lowaffinity to anti-thrombin III exhibited a comparable highanti-metastatic activity, indicating that the heparanase inhibitingactivity of heparin, rather than its anticoagulant activity, plays arole in the anti-metastatic properties of the polysaccharide (12).

Finally, heparanase externally adhered to B16-F1 melanoma cellsincreased the level of lung metastases in C57BL mice as compared tocontrol mice (see U.S. patent application Ser. No. 09/260,037, entitledINTRODUCING A BIOLOGICAL MATERIAL INTO A PATIENT, which is acontinuation in part of U.S. patent application Ser. No. 09/140,888, andis incorporated herein by reference.

Possible Involvement of Heparanase in Tumor Angiogenesis

Fibroblast growth factors are a family of structurally relatedpolypeptides characterized by high affinity to heparin (29). They arehighly mitogenic for vascular endothelial cells and are among the mostpotent inducers of neovascularization (29-30). Basic fibroblast growthfactor (bFGF) has been extracted from a subendothelial ECM produced invitro (31) and from basement membranes of the cornea (32), suggestingthat ECM may serve as a reservoir for bFGF. Immunohistochemical stainingrevealed the localization of bFGF in basement membranes of diversetissues and blood vessels (23). Despite the ubiquitous presence of bFGFin normal tissues, endothelial cell proliferation in these tissues isusually very low, suggesting that bFGF is somehow sequestered from itssite of action. Studies on the interaction of bFGF with ECM revealedthat bFGF binds to HSPG in the ECM and can be released in an active formby HS degrading enzymes (33, 32, 34). It was demonstrated thatheparanase activity expressed by platelets, mast cells, neutrophils, andlymphoma cells is involved in release of active bFGF from ECM andbasement membranes (35), suggesting that heparanase activity may notonly function in cell migration and invasion, but may also elicit anindirect neovascular response. These results suggest that the ECM HSPGprovides a natural storage depot for bFGF and possibly otherheparin-binding growth promoting factors (36,37). Displacement of bFGFfrom its storage within basement membranes and ECM may therefore providea novel mechanism for induction of neovascularization in normal andpathological situations.

Recent studies indicate that heparin and HS are involved in binding ofbFGF to high affinity cell surface receptors and in bFGF cell signaling(38, 39). Moreover, the size of HS required for optimal effect wassimilar to that of HS fragments released by heparanase (40). Similarresults were obtained with vascular endothelial cells growth factor(VEGF) (41), suggesting the operation of a dual receptor mechanisminvolving HS in cell interaction with heparin-binding growth factors. Itis therefore proposed that restriction of endothelial cell growthfactors in ECM prevents their s systemic action on the vascularendothelium, thus maintaining a very low rate of endothelial cellsturnover and vessel growth. On the other hand, release of bFGF fromstorage in ECM as a complex with HS fragment, may elicit localizedendothelial cell proliferation and neovascularization in processes suchas wound healing, inflammation and tumor development (36,37).

Recombinant Heparanase for Screening Purposes

Put together, the accumulated evidences indicate that a reliable andhigh throughput (HTS) screening system for heparanase inhibitingcompounds may be applied to identify and develop non-toxic drugs for thetreatment of cancer and metastasis. Research aimed at identifying anddeveloping inhibitors of heparanase catalytic activity has beenhandicapped by the lack of a consistent and constant source of apurified and highly active heparanase enzyme and of a reliable screeningsystem. Such a HTS system is described in U.S. patent application Ser.No. 09/113,168, which is incorporated herein by reference. To this end,however, methods are required for obtaining high quantities of highlypure and active heparanase, so as to enable to study the kinetics ofheparanase per se and in the presence of potential inhibitors. Therecent cloning, expression and purification of the humanheparanase-encoding gene offer, for the first time, a most appropriateand reliable source of active recombinant enzyme for screening ofanti-heparanase antibodies and compounds which may inhibit the enzymeand hence be applied to identify and develop drugs that may inhibittumor metastasis, autoimmune and inflammatory diseases.

Screening for Specific Inhibitors Using a Combinatorial Library

A new approach aimed at rational drug discovery was recently developedfor screening for specific biological activities. According to the newapproach, a large library of chemically diverged molecules are screenedfor the desired biological activity. The new approach has become aneffective and hence important tool for the discovery of new drugs. Thenew approach is based on “combinatorial” synthesis of a diverse set ofmolecules in which several components predicted to be associated withthe desired biological activity are systematically varied. The advantageof a combinatorial library over the alternative use of natural extractsfor screening for desired biologically active compounds is that all thecomponents comprising the library are known in advance (60).

In combinatorial screening, the number of hits discovered isproportional to the number of molecules tested. This is true even whenknowledge concerning the target is unavailable. The large number ofcompounds, which may reach thousands of compounds tested per day, canonly be screened, provided that a suitable assay involving a highthroughput screening technique, in which laboratory automation androbotics may be applied, exists.

Expression of Heparanase by Cells of the Immune System

Heparanase catalytic activity correlates with the ability of activatedcells of the immune system to leave the circulation and elicit bothinflammatory and autoimmune responses. Interaction of platelets,granulocytes, T and B lymphocytes, macrophages and mast cells with thesubendothelial ECM is associated with degradation of heparan sulfate(HS) by heparanase catalytic activity (10). The enzyme is released fromintracellular compartments (e.g., lysosomes, specific granules) inresponse to various activation signals (e.g., thrombin, calciumionophore, immune complexes, antigens, mitogens), suggesting itsregulated involvement and presence in inflammatory sites and autoimmunelesions. Heparan sulfate degrading enzymes released by platelets andmacrophages are likely to be present in atherosclerotic lesions (42).

Treatment of experimental animals with heparanase alternative substrates(e.g., non-anticoagulant species of low molecular weight heparin)markedly reduced the incidence of experimental autoimmuneencephalomyelitis (EAE), adjuvant arthritis and graft rejection (10, 43)in experimental animals, indicating that heparanase inhibitors may beapplied to inhibit autoimmune and inflammatory diseases (10,43).

The Involvement of Heparanase in Other Physiological Processes and itsPotential Therapeutic Applications

Apart from its involvement in tumor cell metastasis, inflammation andautoimmunity, mammalian heparanase may be applied to modulatebioavailability of heparin-binding growth factors (45); cellularresponses to heparin-binding growth factors (e.g., bFGF, VEGF) andcytokines (IL-8) (44, 41); cell interaction with plasma lipoproteins(49); cellular susceptibility to certain viral and some bacterial andprotozoa infections (45-47); and disintegration of amyloid plaques (48).

Viral Infection

The presence of heparan sulfate on cell surfaces have been shown to bethe principal requirement for the binding of Herpes Simplex (45) andDengue (46) viruses to cells and for subsequent infection of the cells.Removal of the cell surface heparan sulfate by heparanase may thereforeabolish virus infection. In fact, treatment of cells with bacterialheparitinase (degrading heparan sulfate) or heparinase (degradingheparan) reduced the binding of two related animal herpes viruses tocells and rendered the cells at least partially resistant to virusinfection (45). There are some indications that the cell surface heparansulfate is also involved in HIV infection (47).

Neurodegenerative Diseases

Heparan sulfate proteoglycans were identified in the prion proteinamyloid plaques of Genstmann-Straussler Syndrome, Creutzfeldt-Jakobdisease and Scrape (48). Heparanase may disintegrate these amyloidplaques which are also thought to play a role in the pathogenesis ofAlzheimer's disease.

Restenosis and Atherosclerosis

Proliferation of arterial smooth muscle cells (SMCs) in response toendothelial injury and accumulation of cholesterol rich lipoproteins arebasic events in the pathogenesis of atherosclerosis and restenosis (50).Apart from its involvement in SMC proliferation as a low affinityreceptor for heparin-binding growth factors, HS is also involved inlipoprotein binding, retention and uptake (51). It was demonstrated thatHSPG and lipoprotein lipase participate in a novel catabolic pathwaythat may allow substantial cellular and interstitial accumulation ofcholesterol rich lipoproteins (49). The latter pathway is expected to behighly atherogenic by promoting accumulation of apoB and apoE richlipoproteins (e.g., LDL, VLDL, chylomicrons), independent of feed backinhibition by the cellular cholesterol content. Removal of SMC HS byheparanase is therefore expected to inhibit both SMC proliferation andlipid accumulation and thus may halt the progression of restenosis andatherosclerosis.

In summary, Heparanase may thus prove useful for conditions such aswound healing, angiogenesis, restenosis, atherosclerosis, inflammation,neurodegenerative diseases and viral infections. Mammalian heparanasecan be used to neutralize plasma heparin, as a potential replacement ofprotamine. Anti-heparanase antibodies may be applied for immunodetectionand diagnosis of micrometastases, autoimmune lesions and renal failurein biopsy specimens, plasma samples, and body fluids. Common use inbasic research is expected.

ECM Proteases and Their Involvement in Tumor Progression and Metastasis

The cooperation with pericellular proteolysis cascades is required forvascular remodeling during angiogenesis, inflammatory processes, tumorprogression and metastasis. In particular, the invasive processes thatoccur during tumor progression—local invasion, intravasation,extravasation and metastasis formation—involve extracellular matrix(ECM) degradation by proteases.

Four classes of proteases, are known to correlate with malignantphenotype: (i) cysteine proteases including cathepsin B and L; (ii)aspartyl protease cathepsin D; (iii) serine proteases including plasmin,tissue-type plasminogen activator (tPA) and urokinase-type plasminogenactivator (uPA), (iv) Matrix metalloproteinases (MMPs) includingcollagenases, gelatinases A and B (MMP2 and MMP9) and stromelysin(MMP3).

Cathepsins are a family of proteases that are found inside cells innormal physiological conditions. Secretion of cathepsins correlates withvarious pathological conditions, such as arthritis, Alzheimer's diseaseand cancer progression (52).

The lysosomal cystein proteases cthepsin B and L have been suggested toplay a role in tumor cell invasion and spread, either by directlycleaving extracellular matrix proteins or indirectly by activating otherproteases (53).

Cathepsin B was found to have elevated expression levels in cancercells. Furthermore, the intracellular distribution of the proteindiffered between invasive and non-invasive cancer cells. In invasivecells, cathepsin B was found in the plasma membrane, whereas innon-invasive cells it was confined to the lysosomes (56). In human tumorcells cathepsin B was secreted from the cells (53) and was shown todegrade extracellular matrix components (54). Cathepsin B and L havebeen shown to degrade type IV collagen, laminin and fibronectin in vitroat both acid and neutral pH (54). Both enzymes are able to activate theproenzyme form of the urokinase-type plasminogen activator (pro-uPA),which is secreted by tumor cells and can bind to receptors on the tumorcell surface (55). In this cascade mechanism, the lysosomal cysteineproteases may function as effective mediators of tumor associatedproteolysis.

MMPs are a family of zinc dependent endopeptidases. They are secreted asinactive proenzymes and are activated by limited proteolysis (57).During human pregnancy, cytotrophoblasts adopt tumor-like properties:they attach the conceptus to the endometrium by invading the uterus andthey initiate blood flow to the placenta by breaching maternal vessels.Matrix metalloproteinase MMP-9 (a type IV collagenase/gelatinase) wasshown to be upregulated during cytotrophoblast differentiation along theinvasive pathway. Furthermore, it was shown that the activity of thatprotease specified the ability of the cells to degrade ECM components invitro (58).

Large body of evidence suggests that the matrix metalloproteinases MMP-2and MMP-9 play an important role in tumor invasion process (59, 58).

There is clearly a widely recognized need for, and it would be highlyadvantageous to have, genetically modified cells overexpressingrecombinant heparanase or modified species thereof, methods ofoverexpressing recombinant heparanase in cellular systems and methods ofpurifying recombinant heparanase, so as to enable, a search forheparanase inhibitors using a high throughput assay and a combinatorialapproach.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided arecombinant cell comprising a polynucleotide sequence encoding apolypeptide having heparanase catalytic activity, the cell expressingrecombinant heparanase.

According to a further aspect of the present invention, there isprovided a method of obtaining recombinant heparanase comprising thesteps of genetically modifying a cell with an expression vectorincluding a polynucleotide sequence encoding a polypeptide havingheparanase catalytic activity, the cell expressing recombinantheparanase.

According to still further features in the described preferredembodiments the polynucleotide sequence is as set forth in SEQ ID NO:1or a functional part thereof, the part encodes the polypeptide havingthe heparanase catalytic activity.

According to still further features in the described preferredembodiments the polypeptide includes an amino acid sequence as set forthin SEQ ID NO:2 or a functional part thereof having the heparanasecatalytic activity. The functional part may be the result of eithergenetic engineering natural processing by the transduced cell.

According to still further features in the described preferredembodiments the polynucleotide sequence is selected from the groupconsisting of double stranded DNA, single stranded DNA and RNA.

According to still further features in the described preferredembodiments the cell is a bacterial cell.

According to still further features in the described preferredembodiments the cell is E. coli.

According to still further features in the described preferredembodiments the cell is an animal cell.

According to still further features in the described preferredembodiments the animal cell is an insect cell.

According to still further features in the described preferredembodiments the insect cell is selected from the group consisting ofHigh five and Sf21 cells.

According to still further features in the described preferredembodiments the animal cell is a mammalian cell, selected, for example,from the group consisting of a Chinese hamster ovary cell line (CHO),baby hamster kidney cells (BHK21), Namalwa cells, Dauidi cells, Rajicells, Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-Hep1cells, MDCK₁ cells, MDBK₁ cells, Vero cells, Cos cells, CV-1 cells,NIH3T3 cells, L929 cells and BLG cells (mouse melanoma).

According to still further features in the described preferredembodiments the cell is a yeast cell.

According to still further features in the described preferredembodiments the yeast cell is a methylotrophic yeast.

According to still further features in the described preferredembodiments the yeast cell is selected from the group consisting ofPichia pastoris, Hansenula polymorpha and Saccharomyces cerevisiae.

According to still further features in the described preferredembodiments the heparanase is human recombinant heparanase.

According to still further features in the described preferredembodiments the polynucleotide sequence is integrated in the cell'sgenome rendering the cell a stably transduced.

According to still further features in the described preferredembodiments the polynucleotide sequence is external to the cell'sgenome, rendering the cell transiently transduced.

According to still further features in the described preferredembodiments the polynucleotide sequence forms a part of a viral genomeinfective to the cell, be it bacterial or animal cell.

According to still further features in the described preferredembodiments the polynucleotide sequence encodes, in addition, a signalpeptide for protein secretion.

According to still further features in the described preferredembodiments the method further comprising the step of subjecting thecell to a substance which induces secretion into the growth medium ofsecretable proteins, thereby inducing secretion of the recombinantheparanase into the growth medium.

According to still further features in the described preferredembodiments the substance is selected from the group consisting ofthrombin, calcium ionophores, immune complexes, antigens and mitogens.

According to still further features in the described preferredembodiments the calcium ionophore is calcimycin (A23187).

According to still further features in the described preferredembodiments the substance is phorbol 12-myristate 13-acetate (PMA).

According to still further features in the described preferredembodiments the method further comprising the step of purifying therecombinant heparanase.

According to still further features in the described preferredembodiments the purification is effected in part by an ion exchange(e.g., Source-S) column.

According to still further features in the described preferredembodiments the purification is from the cell.

According to still further features in the described preferredembodiments the purification is from a growth medium in which the cellis grown.

According to still further features in the described preferredembodiments the cell is grown in a large biotechnological scale of atleast half a liter growth medium.

According to another aspect of the present invention provided is amethod of purifying a recombinant heparanase from overexpressing cellsor growth medium comprising the steps of adsorbing the recombinantheparanase on an ion exchange (e.g., Source-S) column under low saltconditions, washing the column with low salt solution thereby elutingother proteins, and eluting the recombinant heparanase from the columnby a salt gradient or a higher salt concentration.

According to a further aspect of the present invention there is provideda method of activating a heparanase enzyme comprising the step ofdigesting the heparanase enzyme by a protease.

According to still further features in the described preferredembodiments the protease is selected from the group consisting of acysteine protease, an aspartyl protease, a serine protease and ameatlloproteinase.

According to still further features in the described preferredembodiments the step of digesting the heparanase enzyme by a protease iseffected at a pH in which the protease is active, preferably mostactive.

According to a further aspect of the present invention there is provideda method of in vivo inhibition of proteolytic processing of heparanasecomprising the step of in vivo administering a protease inhibitor.

According to still further features in the described preferredembodiments the protease inhibitor is selected from the group consistingof a cysteine protease inhibitor, an aspartyl protease inhibitor, aserine protease inhibitor and a meatlloproteinase inhibitor.

According to a further aspect of the present invention there is provideda nucleic acid construct comprising a first nucleic acid segmentencoding for an upstream portion of heparanase, a second, in frame,nucleic acid sequence encoding a recognition and cleavage sequence of aprotease and a third, in frame, nucleic acid sequence encoding for adownstream portion of heparanase, wherein the second nucleic acidsequence is in between the first nucleic acid sequence and the thirdnucleic acid sequence.

According to still further features in the described preferredembodiments the protease is selected having no recognition and cleavagesequences in the upstream and the downstream portions of heparanase.

According to still further features in the described preferredembodiments the third nucleic acid sequence encodes for a catalyticallyactive heparanase when correctly folded.

According to a further aspect of the present invention there is provideda precursor heparanase protein comprising an upstream portion ofheparanase, a mid portion of a recognition and cleavage sequence of aprotease and a downstream portion of heparanase, wherein the protease isselected having no recognition and cleavage sequences in the upstreamand the downstream portions of heparanase.

According to a further aspect of the present invention there is provideda heparanase protein resulting by digesting the precursor heparanaseprotein described herein.

According to a further aspect of the present invention there is provideda method of obtaining a homogeneously processed, active heparanase, themethod comprising the steps of (a) expressing the precursor heparanaseprotein in a cell which secretes the precursor heparanase protein intothe growth medium to obtain a conditioned growth medium, the precursorheparanase protein including an upstream portion of heparanase, a midportion of a recognition and cleavage sequence of a protease and adownstream portion of heparanase, wherein the protease is selectedhaving no recognition and cleavage sequences in the upstream and thedownstream portions of heparanase; (b) treating the precursor heparanaseprotein with the protease; and (c) purifying a proteolytic heparanaseproduct having heparanase catalytic activity.

According to a further aspect of the present invention there is providedan antibody comprising an immunoglobulin elicited against recombinantnative heparanase.

According to a further aspect of the present invention there is providedan affinity substrate comprising a solid matrix and an immunoglobulinelicited against recombinant native heparanase being immobilizedthereto.

According to a further aspect of the present invention there is provideda method of affinity purifying heparanase comprising the steps of (a)loading a heparanase preparation on an affinity substrate including asolid matrix and an immunoglobulin elicited against recombinant nativeheparanase being immobilized thereto; (b) washing the affinitysubstrate; and (c) eluting heparanase molecules being adsorbed on theaffinity substrate via the immunoglobulin.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing cells and methods forexpressing recombinant heparanase, methods for purifying the recombinantheparanase and modified heparanase precursor species which can beprocessed to yield highly active heparanase. Other features andadvantages of the various embodiments of the present invention arefurther addressed hereinunder.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention herein described, by way of example only, with referenceto the accompanying drawings, wherein:

FIG. 1 demonstrates the expression of recombinant heparanase in E. coliBL21(DE3)pLysS cells. Insoluble fractions of induced E. coli cellscontaining expression constructs for heparanase were analyzed on 10%SDS-PAGE. Following electrophoresis the gel was stained with commassieblue. Lane 1—cells transformed with pRSET (negative control), lanes 2and 3—cells transformed with pRSEThpaSl (two different colonies).Molecular size in kDa is shown to the left (Prestained SDS-PAGEstandards, Bio-Rad, CA).

FIG. 2 is a schematic presentation of the expression vectorpPIC3.5K-Sheparanase. Relative positions of some restriction enzymes andgenes are indicated. For the construction and utilities ofpPIC3.5K-Sheparanase, see Example 2 in the Examples section below.

FIG. 3 is a schematic presentation of the expression vector pPIC9K-PP2.Positions of some restriction enzymes and genes are indicated. For theconstruction and utilities of pPIC3.5K-Sheparanase, see Example 2 in theExamples section below.

FIG. 4 demonstrates the secretion of human heparanase by transformedPichia pastoris yeast cells. Western blot analysis using a rabbitanti-heparanase polyclonal antibody (disclosed in U.S. patentapplication Ser. No. 09/071,618, which is incorporated by reference asif fully set forth herein) was performed on culture supernatants ofdifferent transformants (with and without selection for G-418resistance). Lane 1—pPIC3.5K-Sheparanase transformant, lane 2—pPIC3.5Ktransformant (negative control), lanes 3-6, transformants selected on 4mg/ml of G-418. Molecular size is shown on the right as was determinedusing prestained SDS-PAGE standards, BioRad, CA.

FIGS. 5a-e are schematic presentations of heparanase expression vectorsadapted to direct heparanase expression in animal cells. hpa containingplasmids pShpa, pShpaCdhfr, pS1hpa, pS2hpa and pChpa are of 5374 bp,7090 bp, 6868 bp, 6892 bp and 6540 bp, respectively. SV40 prom—SV40early promoter, CMV prom—Citomegalovirus promoter, dhfr—mousedihydrofolate reductase gene, PPT—preprotrypsin signal peptide,hpa—heparanase cDNA sequence, hpa′ and hpa″—truncated hpa sequences.

FIGS. 6a-b show Western blot analysis of hpa transfected cells. Cellextracts (40 μg of CHO cells or 8 μg of 293 cells) were separated on4-20% gradient SDS-PAGE and transferred to PVDF membranes. Detection ofhpa gene products was performed with a rabbit anti-heparanase polyclonalantibody (disclosed in U.S. patent application Ser. No. 09/071,739)followed by ECL detection (Amersham, UK). FIG. 6a—CHO stable cellularclones (lanes 1-3) and transiently transfected 293 human cells (lane 4).FIG. 6b—Mock transfected CHO cells (lane 3), CHO cells performing stableor transient expression (lanes 1 and 2, respectively). Molecular size inkDa is shown to the right, as was determined using prestained SDS-PAGEstandards, Bio-Rad, CA.

FIGS. 7a-b demonstrate recombinant heparanase secretion induced bycalcium ionophore and PMA. Cells of a stable CHO clone (2TT1) wereinduced with either calcium ionophore (FIG. 7a) or PMA (FIG. 7b).Condition media were collected and 20 ml loaded on SDS polyacrylamidegel followed by Western blot analysis with a rabbit anti-heparanasepolyclonal antibody (disclosed in U.S. patent application Ser. No.09/071,739) followed by ECL detection (Amersham, UK). Molecular size inkDa is shown on the right, as was determined using prestained SDS-PAGEstandards, Bio-Rad, CA.

FIG. 7c demonstrates recombinant heparanase secretion by human 293cells. Conditioned media of human 293 cells transfected with pS1hpa(lanes 3 and 4), pS2hpa (lanes 5 and 6) or control, untransfected cells(lanes 1 and 2), were loaded on a denaturative 4-20% polyacrylamide gel(lanes 1, 3 and 5), or 5 fold concentrated by 10 kDa ultrafiltrationtube (Intersep U.K.) (lanes 4 and 6). Heparanase was detected by Westernblot analysis with a rabbit anti-heparanase polyclonal antibody(disclosed in U.S. patent application Ser. No. 09/071,618) followed byECL detection (Amersham, UK). Molecular size in kDa is shown on theleft, as was determined using prestained SDS-PAGE standards, Bio-Rad,CA.

FIG. 8a demonstrates heparanase activity as expressed by the ability todegrade heparin. Following overnight incubation with 50 mlunconcentrated (lanes 3, 6), 20×concentrated (lanes 4 and 7) or40×concentrated (lanes 5 and 8) conditioned media, from untreated (lanes3-5) versus treated (lanes 6-8, 2 hours of incubation with 1 mg/mlcalcium ionophore) stable clones, samples were electrophoreticallyseparated on 7.5% polyacrylamide gel. Undegraded and degraded (bypurified natural human heparanase) controls are shown in lanes 1 and 2respectively.

FIGS. 8b-c demonstrate recombinant heparanase activity followingsecretion induced by calcium ionophore as determined by the soluble³⁵S-ECM degradation assay. 8 b—the heparanase activity in one mluntreated conditioned media (c60), compared to one ml conditioned mediatreated with 100 ng/ml calcium ionophore for 24 hours (p70) from stableCHO clones was determined by the soluble ³⁵S-ECM degradation assay. 8c—the heparanase activity in one ml untreated conditioned media (c45),compared to one ml conditioned media treated with 1 mg/ml calciumionophore for two hours (p52) from stable CHO clones was determined bythe soluble ³⁵S-ECM degradation assay. Degraded substrates shift to theright.

FIGS. 8d-g show the relative heparanase activity of p70 and p52 (seeFIGS. 8b-c) by comparing the ability of diluted (×2, ×4 or ×8)conditioned media to degrade ³⁵S-ECM.

FIG. 9 demonstrates glucose consumption record of heparanase producingcells in a large scale, 0.5 liters, Spinner-Basket bioreactor.

FIG. 10 demonstrates degradation of soluble sulfate labeled HSPGsubstrate by lysates of High five cells infected with pFhpa2 virus.Lysates of High five cells that were infected with pFhpa2 virus () orcontrol pF2 virus (□) were incubated (18 h, 37° C.) with sulfate labeledECM-derived soluble HSPG (peak I). The incubation medium was thensubjected to gel filtration on Sepharose 6B. Low molecular weight HSdegradation fragments (peak II) were produced only during incubationwith the pFhpa2 infected cells, but there was no degradation of the HSPGsubstrate () by lysates of pF2 infected cells.

FIGS. 11a-b demonstrate degradation of soluble sulfate labeled HSPGsubstrate by the growth medium of pFhpa2 and pFhpa4 infected cells.Culture media of High five cells infected with pFhpa2 (11 a) or pFhpa4(11 b) viruses (), or with control viruses (□) were incubated (18 h,37° C.) with sulfate labeled ECM-derived soluble HSPG (peak I, ). Theincubation media were then subjected to gel filtration on Sepharose 6B.Low molecular weight HS degradation fragments (peak II) were producedonly during incubation with the hpa gene containing viruses. There wasno degradation of the HSPG substrate by the growth medium of cellsinfected with control viruses.

FIG. 12 presents size fractionation of heparanase activity expressed bypFhpa2 infected cells. Growth medium of pFhpa2 infected High five cellswas applied onto a 50 kDa cut-off membrane. Heparanase activity(conversion of the peak I substrate, () into peak II HS degradationfragments) was found in the high (>50 kDa) (), but not low (<50 kDa)(◯) molecular weight compartment.

FIGS. 13a-b demonstrate the effect of heparin on heparanase activityexpressed by pFhpa2 and pFhpa4 infected High five cells. Culture mediaof pFhpa2 (13 a) and pFhpa4 (13 b) infected High five cells wereincubated (18 h, 37° C.) with sulfate labeled ECM-derived soluble HSPG(peak I, ) in the absence () or presence (Δ) of 10 μg/ml heparin.Production of low molecular weight HS degradation fragments wascompletely abolished in the presence of heparin, a potent competitor forheparanase activity.

FIGS. 14a-b demonstrate degradation of sulfate labeled intact ECM byvirus infected High five and Sf21 cells. High five (14 a) and Sf21 (14b) cells were plated on sulfate labeled ECM and infected (48 h, 28° C.)with pFhpa4 () or control pFl (□) viruses. Control non-infected Sf21cells (R) were plated on the labeled ECM as well. The pH of the culturedmedium was adjusted to 6.0-6.2 followed by 24 h incubation at 37° C.Sulfate labeled material released into the incubation medium wasanalyzed by gel filtration on Sepharose 6B. HS degradation fragmentswere produced only by cells infected with the hpa containing virus.

FIGS. 15a-b demonstrate degradation of sulfate labeled intact ECM byvirus infected cells. High five (15 a) and Sf21 (15 b) cells were platedon sulfate labeled ECM and infected (48 h, 28° C.) with pFhpa4 () orcontrol pF1 (□) viruses. Control non-infected Sf21 cells (R) were platedon labeled ECM as well. The pH of the cultured medium was adjusted to6.0-6.2, followed by 48 h incubation at 28° C. Sulfate labeleddegradation fragments released into the incubation medium was analyzedby gel filtration on Sepharose 6B. HS degradation fragments wereproduced only by cells infected with the hpa containing virus.

FIGS. 16a-b demonstrate degradation of sulfate labeled intact ECM by thegrowth medium of pFhpa4 infected cells. Culture media of High five (16a) and Sf21 (16 b) cells that were infected with pFhpa4 () or controlpF1 (□) viruses were incubated (48 h, 37° C., pH 6.0) with intactsulfate labeled ECM. The ECM was also incubated with the growth mediumof control non-infected Sf21 cells (R). Sulfate labeled materialreleased into the reaction mixture was subjected to gel filtrationanalysis. Heparanase activity was detected only in the growth medium ofpFhpa4 infected cells.

FIGS. 17a-b demonstrate the effect of heparin on heparanase activity inthe growth medium of pFhpa4 infected cells. Sulfate labeled ECM wasincubated (24 h, 37° C., pH 6.0) with growth medium of pFhpa4 infectedHigh five (17 a) and Sf21 (17 b) cells in the absence () or presence(V) of 10 μg/ml heparin. Sulfate labeled material released into theincubation medium was subjected to gel filtration on Sepharose 6B.Heparanase activity (production of peak II HS degradation fragments) wascompletely inhibited in the presence of heparin.

FIG. 18 demonstrate the purification of recombinant heparanase by aSource-S column. Lanes 1-14, 40 ml of fractions 1-14 eluted from aSource-S column. Samples were analyzed on 8-16% gradient SDS-PAGE. Gelwas stained with commassie blue.

FIG. 19 demonstrate Western blot analysis of fractions 1-14 of FIG. 18.Fractions 1-14 eluted from a Source-S column were analyzed followingblotting onto nitrocellulose membrane with a rabbit antiheparanasepolyclonal antibody (disclosed in U.S. patent application Ser. No.09/071,739) followed by ECL detection (Amersham, UK).

FIG. 20 is a schematic presentation of plasmid pCdhfr that contains themouse dhfr gene under CMV promoter regulation. This vector does notexpress heparanase and serves as negative control.

FIG. 21a demonstrates the production of heparanase in pS1 hpatransfected BHK21 cells. Cell extracts (2×10⁵ BHK21 cells) wereseparated on 8-16% gradient SDS-PAGE and transferred to PVDF membranes.Detection of hpa gene products was performed with a mouseanti-heparanase monoclonal antibody No. HP-117 (disclosed in U.S. patentapplication Ser. No. 09/071,739) followed by ECL detection (Amersham,UK). Molecular size in kDa is shown to the right, as was determinedusing prestained SDS-PAGE standards, Bio-Rad, CA. Lane 1 pS1hpatransfected BHK21 cells. Lane 2 control, pCdhfr transfected, BHK21cells.

FIG. 21b demonstrates heparanase activity in human 293 cell extract.Cells were collected and concentrated by centrifugation (2750×g for 5min). The pellets were passed through three cycles of 5 minutes freezingin liquid nitrogen and thawing at 37° C. Cell lysate was centrifuged for15 minutes at 3000×g, and the supernatant was collected for analysis.Increasing amounts of supernatant, between 0 and 5 μg protein per assaywere assayed using the DMB activity assay described herein (see alsoU.S. patent application Ser. No. 09/113,168).

FIG. 22a demonstrates recombinant heparanase constitutive secretion byCHO cells transfected with pS1hpa (clone S1PPT-8). Conditioned media (20μl) of untreated cells (lane 2), mock treated cells (lane 3) and calciumionophore treated cells (0.1 μg/ml for 24 hours; lane 4) wereelectrophoresed next to a cellular extract of 1×10⁵ cells from clone2TT1 (CHO cells transfected with pShpaCdhfr, lane 1). Samples wereseparated on a 4-20% gradient SDS-PAGE, followed by Western blotanalysis with a rabbit anti-heparanase polyclonal antibody (disclosed inU.S. patent application Ser. No. 09/071,739) and by ECL detection(Amersham, UK). 20 Molecular size in kDa is shown on the right, as wasdetermined using prestained SDS-PAGE standards, Bio-Rad, CA.

FIG. 22b demonstrates recombinant heparanase constitutive secretion byCHO cells transfected with pShpaCdhfr (2TT1 clones). Conditioned media(150 μl, concentrated by 10 kDa ultrafiltration tube) of 2TT1-2 clone(lane 2) and of clone 2TT1-8 (lane 3) were electrophoresed next to acellular extract of 1×10⁵ cells from clone 2TT1 (lane 1). Samples wereseparated on a 4-20% gradient SDS-PAGE, followed by Western blotanalysis with a rabbit anti-heparanase polyclonal antibody (disclosed inU.S. patent application Ser. No. 09/071,739) and by ECL detection(Amersham, UK). Molecular size in kDa is shown on the right, as wasdetermined using prestained SDS-PAGE standards, Bio-Rad, CA.

FIG. 23a demonstrates purification of recombinant heparanase from amammalian cellular extract by ion exchange chromatography. 2TT1-8 CHOcells (1×10⁸) were extracted in 2.5 ml of 10 mM phosphate citrate bufferpH 5.4. The extract was centrifuged at 2750×g for 5 minutes and thesupernatant was collected for heparanase enzyme purification using acation exchange chromatography column. The chromatography column (mono-SHR 5/5, Pharmacia Biotech) was equilibrated with 20 mM sodium phosphatebuffer, pH 6.8, and the mixture was loaded atop thereof. Proteins wereeluted from the column using a linear gradient of 0 to 1 M sodiumchloride in 20 mM sodium phosphate buffer, pH 6.8. The gradient wascarried out in 20 column volumes at a flow rate of one ml per minute.The elution of proteins was monitored at 214 nm and fractions of 1 mleach were collected, starting with the first fraction (1) which waseluted after 13 minutes and which is identified by the arrowhead mark.

FIG. 23b demonstrates the presence of immunologically active recombinantheparanase in the mammalian cellular extract. An aliquot from eachfraction that was collected was analyzed for the presence of theheparanase enzyme by Western blot analysis. 20 μl from each fraction,numbered 1-26, were separated on a 4-20% SDS-PAGE. The proteins weretransferred from the gel to a PVDF membrane and were detected with amonoclonal antibody No. HP-117 (disclosed in U.S. patent applicationSer. No. 09/071,739) followed by ECL detection (Amersham, UK). Molecularsize in kDa is shown to the right, as was determined using SDS-PAGEstandards (M). St—a purified recombinant heparanase enzyme from CHOcells.

FIG. 23c demonstrates the presence of catalytically active recombinantheparanase in mammalian cellular extract fractions. An aliquot (30 μl)from each fraction that was collected was analyzed for heparanaseactivity by the DMB assay. Load—extract prior to purification. 5-7 and16-26 correspond to fraction Nos.

FIG. 23d demonstrates a heparanase dose response. Increasing amountsfrom fraction No. 20, which exhibited the highest activity using the DMBassay (FIG. 23c), were analyzed for heparanase activity using thetetrazolium assay, as disclosed in U.S. patent application Ser. No.09/113,168.

FIG. 24a demonstrates the purification of heparanase from a mammaliancellular extract by an affinity column. A cellular extract from CHO2TT1-8 cells was loaded on an affinity column containing antibodieselicited against native (non-denatured) recombinant heparanase. Westernblot analysis of different fractions (1-6) using a monoclonal antibodyNo. HP-117 (disclosed in U.S. patent application Ser. No. 09/071,739)followed by ECL detection (Amersham, UK) is shown. Molecular size in kDais shown to the left, as was determined using SDS-PAGE standards (M).A—recombinant heparanase enzyme purified from CHO 2TT1-8 cells on mono-Scolumn; B—extract of 2TT1-8 cells; C—unbound, flow through proteins; andD—wash fraction proteins.

FIG. 24b demonstrates the purification of heparanase from a mammaliancellular extract by an affinity column. A cellular extract from CHO2TT1-8 cells was loaded on an affinity column containing antibodieselicited against native (non-denatured) recombinant heparanase.Heparanase activity in affinity column fraction Nos. 1-9 was determinedusing the DMB assay. Load—extract prior to purification; C—unbound, flowthrough proteins; and D—wash fraction proteins.

FIGS. 25a-b demonstrates proteolytic processing of heparanase frominsect cells conditioned medium by protease impurities. FIG. 25a shows aWestern blot analysis of heparanase, following processing of the enzymeexpressed in insect cells. Heparanase expressed in insect cells,partially purified on a Source-S column, was incubated for one week at4° C. in either, 20 mM phosphate citrate buffer pH 7, containing 5% PEG300 (lane A), 20 mM phosphate citrate buffer pH 4, containing 5% PEG 300and 1×protease inhibitors cocktail (Boehringer Mannheim, Cat. No.1836170, lane B), or 20 mM phosphate citrate buffer pH 4, containing 5%PEG 300 (lane C). M—Molecular weight markers (NEB Cat. No. 7708S). FIG.25b shows the results of DMB heparanase activity assays for theproteins.

FIGS. 25c-d demonstrate the effect of a panel of protease inhibitors onproteolytic processing and activation of heparanase expressed in insectcells. Heparanase expressed in insect cells, partially purified on aSource-S column, was incubated for one week at 4° C. in 20 mM phosphatecitrate buffer, pH 4, containing 5% PEG 300 and one of the differentprotease inhibitors: A—antipain; B—bestatin; C—chymostatin; D—E-64;E—leupeptin; F—pepstatin; G—phosphoramidon; H—EDTA; I—aprotinin. Thetreated samples were either subjected to western blot analysis (FIG.25c) or to heparanase DMB activity assay (FIG. 25d). J—positive control,incubated in the absence of a protease inhibitor at pH 4; K—negativecontrol, heparanase incubated with the same buffer at pH 7. M—Molecularweight marker (NEB Cat. No. 7708S).

FIG. 26a demonstrates proteolytic processing of heparanase secreted frominsect cells by trypsin. 10 μg of heparanase, expressed in insect cells,and partially purified on a Source-S column, was incubated withincreasing concentrations of trypsin (0, 1.5, 5, 10, 15 units/test, Cat.No. T-8642, Sigma USA) for 10 minutes at 25° C. Following incubation,reaction tubes were placed on ice and 1.7 μg/ml aprotinin (trypsininhibitor) was added. Activity was determined using the DMB assay.

FIG. 26b demonstrates a Western blot analysis of heparanase followingtrypsin treatment. 10 μg of heparanase, expressed in insect cells, andpartially purified on a Source-S column, was incubated without (lane 1)or with 150 or 500 units of trypsin (lanes 2 and 3, respectively). Aprocessed heparanase sample treated as described in FIGS. 25a-b, lanes J(lane 4), and heparanase from a CHO 2TT1 cell extract (lane 5) served ascontrols.

FIG. 27 proteolytic processing of heparanase secreted from CHO cells bytrypsin. Conditioned medium of CHO cells transfected with pS1hpa (cloneS1PPT-8) that secrete heparanase in a constitutive manner was subjectedto proteolysis by trypsin. Unpurified CHO conditioned medium containingheparanase (0.5 μg heparanase per reaction) in 20 mM phosphate buffer,pH 6.8, was incubated with 0, 1.5, 15 or 150 units of trypsin for 10minutes, at 37° C. Reactions were stopped by transferring the reactiontubes into ice and adding 2 μg/ml aprotinin. Tryptic digest productswere assayed for heparanase activity using the DMB assay.

FIGS. 28a-b demonstrates proteolytic processing of p70-bac heparanase bycathepsin L. Partially purified heparanase from insect cells (10 μg) wassubjected to proteolysis by 1.6 mU cathepsin L (Cat. No. 219412,Calbiochem) for 3 hours, at 30° C., in 20 mM citrate-phosphate buffer,pH 5.4. Heparanase catalytic activity and immunoreactivity before (1)and after (2) processing with cathepsin L as were determined using theDMB heparanase activity assay and Western blot analysis with monoclonalantibody No. HP-117 (disclosed in U.S. patent application Ser. No.09/071,739) followed by ECL detection (Amersham, UK), FIGS. 28a-b,respectively.

FIG. 29a demonstrates a hydropathy plot of SEQ ID NO:2 predicted forheparanase as calculated by the Kyte-Doolittle method for calculatinghydrophilicity, using the Wisconsin University GCG DNA analysissoftware. I and II point at peaks of most hydrophilic regions of theenzyme.

FIG. 29b is a schematic depiction of modified heparanase species(pre-p56′ and pre-p52′) that contain a unique protease recognition andcleavage sequence of factor Xa—Ile-Glu-Gly-Arg↓—or ofenterokinase—Asp-Asp-Asp-Asp-Lys↓ (shaded regions, located between aminoacids 119 and 120 or 157 and 158 of the heparanase enzyme depicted inSEQ ID NO:2, which acids are located within peaks I and II,respectively, of FIG. 29a) which enable proteolytic processing by therespective proteases to obtain homogeneously processed and highly activeheparanase species (p56′ and p52′, respectively).

FIG. 29c is a schematic depiction of the steps in constructing nucleicacid constructs harboring a unique protease recognition and cleavagesequence of factor Xa—Ile-Glu-Gly-Arg↓—or ofenterokinase—Asp-Asp-Asp-Asp-Lys↓.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of genetically modified cells overexpressingrecombinant heparanase and of methods for overexpressing recombinantheparanase in cellular systems, which can be used to obtain purifiedrecombinant heparanase in large quantities. Specifically, the presentinvention can be used to provide a scheme for biotechnological largescale recombinant heparanase production. The invention further relatesto the activation of heparanase precursors by proteolysis and further tomethods of in vivo inhibition of heparanase activity.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

In one aspect, the present invention provides a genetically modifiedcell transduced with a polynucleotide sequence encoding a polypeptidehaving heparanase catalytic activity, designed to direct expression ofrecombinant heparanase by the cell.

In another aspect, the present invention provides a method of obtainingrecombinant heparanase by genetically modifying a cell with anexpression vector including a polynucleotide sequence encoding apolypeptide having heparanase catalytic activity, designed to directexpression of recombinant heparanase by the cell.

As used herein in the specification and in the claims section below, thephrase “genetically modified cell” refers to a cell that includes arecombinant gene. As further detailed below the cell may be a eukaryoticor prokaryotic cell.

As used herein in the specification and in the claims section below, theterm “transduced” refers to the result of a process of inserting nucleicacids into cells. The insertion may, for example, be effected bytransformation, viral infection, injection, transfection, genebombardment, electroporation or any other means effective in introducingnucleic acids into cells. Following transduction the nucleic acid iseither integrated in all or part, to the cell's genome (DNA), or remainsexternal to the cell's genome, thereby providing stably transduced ortransiently transduced cells.

As used herein in the specification and in the claims section below, thephrase “polynucleotide sequence” also means a nucleic acid sequence,typically a DNA sequence.

As used herein in the specification and in the claims section below, theterm “polypeptide” also means a protein.

As used herein in the specification and in the claims section below, thephrase “heparanase catalytic activity” refers to an animalendoglycosidase hydrolyzing activity which is specific for heparin orheparan sulfate proteoglycan substrates, as opposed to the activity ofbacterial enzymes (heparinase I, II and III) which degrade heparin orheparan sulfate by means of β-elimination.

As used herein in the specification and in the claims section below, theterm “expression” refers to the processes executed by cells whileproducing and/or secreting proteins, including where applicable, but notlimited to, for example, transcription, translation, folding and posttranslational modification and processing.

As used herein in the specification and in the claims section below, theterms “vector” and “construct” are interchangeably used herein and referto any vehicle suitable for genetically modifying cells, including, butnot limited to, viruses (e.g., bacoluvirus), phages, plasmids,phagemids, bacmids, cosmids, artificial chromosomes and the like.

As used herein in the specification and in the claims section below, thephrase “a polynucleotide sequence encoding a polypeptide havingheparanase catalytic activity” refers to the potential of thepolypeptide to have heparanase catalytic activity when correctly folded.Thus, this phrase refers to any catalytically active or inactiveconformant of a polypeptide which may acquire at least one activeconformation having heparanase catalytic activity.

According to a preferred embodiment of the present invention, thepolynucleotide sequence is as set forth in SEQ ID NO:1 or a functionalpart thereof. The functional part encodes a polypeptide havingheparanase catalytic activity. However, the scope of the presentinvention is not limited to SEQ ID NO:1 or a functional part thereof, asnatural and man made innocuous variations thereof (e.g., mutations, suchas point mutations) may also encode a protein having heparanasecatalytic activity. Furthermore, it is shown hereinunder that a 52 kDa(formerly referred to as 45-50 kDa) protein, naturally processed from a70 kDa (formerly referred to as 60 or 60-70 kDa) protein encoded by SEQID NO:1, has heparanase catalytic activity. The polynucleotide sequencemay be a cDNA, a genomic DNA and a composite DNA (including at least oneintron derived from heparanase or any other gene) as further detailed inU.S. patent application Ser. No. 09/071,618, entitled POLYNUCLEOTIDEENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF SAMEIN GENETICALLY MODIFIED CELLS, which is incorporated herein byreference. Similarly it can be derived from any animal includingmammalians and avians because, as shown in U.S. patent application Ser.No. 09/258,892, heparanase sequences derived from species other thanhuman beings are readily hybridizeable with the human sequence, allowingfor isolation of such sequences by methods known in the art.

The functional part may be either man induced by genetic engineering orpost translation artificial processing (e.g., by a protease) ornaturally processed, depending on the cellular system employed.

According to another preferred embodiment of the present invention, thepolypeptide includes an amino acid sequence as set forth in SEQ ID NO:2or a functional part thereof having heparanase catalytic activity.However, the scope of the present invention is not limited to SEQ IDNO:2 or a functional part thereof, as natural and man made innocuousvariations thereof (e.g., mutations, such single amino acidsubstitution) may also have heparanase catalytic activity. Polypeptidescorresponding to species other than human and having heparanasecatalytic activity are also within the scope of the present invention.

As used herein in the specification and in the claims section below, theterm “functional part thereof” refers to a part of a nucleic acidsequence which encodes a polypeptide having heparanase catalyticactivity or a part of a polypeptide sequence having heparanase catalyticactivity.

In this context, it is important to remember that in many casestruncated or naturally processed polypeptides exhibit a catalyticactivity similar to that of the natural polypeptide of the preprocessedpolypeptide, respectively. Apparently, in many cases, not all of theamino acids of a protein are essential for its catalytic function, somemay be responsible for other features, such as secretion, stability,interaction with other macromolecules, etc., whereas other may bereplaced without affecting activity to a great extent. In many cases theprocessed protein exerts higher catalytic activity as compared with itsunprocessed counterpart.

According to yet another preferred embodiment of the present invention,the polynucleotide sequence is selected from the group consisting ofdouble stranded DNA, single stranded DNA and RNA.

According to still another preferred embodiment of the presentinvention, the cell is a bacterial cell, preferably E. coli.

According to a preferred embodiment of the present invention, the cellis an animal cell.

The animal cell may be a mammalian cell, such as, but not limited to,Chinese hamster ovary cell line (CHO), baby hamster kidney cells(BHK21), Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Helacells, Ehrlich's ascites cells, Sk-Hep1 cells, MDCK₁ cells, MDBK₁ cells,Vero cells, Cos cells, CV-1 cells, NIH3T3 cells, L929 cells or BLG cells(mouse melanoma).

Alternatively, the animal cell may be a mammalian cell, such as, but notlimited to, High five or Sf21.

According to another preferred embodiment of the present invention, thecell is a yeast cell, preferably a methylotrophic yeast, such as, butnot limited to, Pichia pastoris and Hansenula polymorpha. Anotherpreferred yeast is Saccharomyces cerevisiae.

The specified bacterial, yeast and animal cells are of specificadvantage and interest since they are widely used in large scalebiotechnological production of proteins and therefore knowledge hasaccumulated with respect to their large scale propagation, maintenanceand with respect to recombinant protein purification therefrom.

According to another preferred embodiment of the present invention, therecombinant heparanase is human recombinant heparanase.

According to another preferred embodiment of the present invention, thepolynucleotide sequence encodes, in addition, a signal peptide forprotein secretion. The signal peptide may be the natural signal peptideof heparanase or any other suitable signal peptide, one non-limitingexample is given under the Examples section hereinunder. The signalpeptide sequence is fused downstream of and in frame with the heparanasesequence.

According to yet another preferred embodiment of the present invention,the method is further effected by purifying the recombinant heparanase.As further detailed hereinunder efficient purification (e.g., 90%purified) of recombinant heparanase may be effected by a single step ionexchange (e.g., Source-S) column.

The purification may be from the cells themselves. To this end the cellsare collected, e.g., by centrifugation, homogenated and the recombinantheparanase is purified from the homogenate. If the recombinantheparanase is secreted by the cells to the growth medium, thenpurification is preferably from the growth medium itself.

According to yet another preferred embodiment of the present invention,the method further includes a step of subjecting the cell to a substancewhich induces secretion into the growth medium of secretable proteins,thereby inducing secretion of the recombinant heparanase into the growthmedium. Preferably, the substance is selected from the group consistingof thrombin, calcium ionophores, immune complexes, antigens andmitogens, all are known to induce secretion of native heparanase fromexpressing cells. As shown in the Examples section below, the calciumionophore calcimycin (A23187) and phorbol 12-myristate 13-acetate, areeffective in inducing secretion of recombinant heparanase fromtransduced cells into their media.

According to yet another preferred embodiment of the present invention,the cell is grown to a large biotechnological scale of at least half aliter, preferably at least 5, 7 or 35 liters of growth medium, in abioreactor, such as but not limited to, Spinner-Basket bioreactor.

Further according to the present invention there is provided a method ofpurifying a recombinant heparanase from overexpressing cells or growthmedium in which they grow by adsorbing the recombinant heparanase on aSource-S column under low salt conditions (e.g., about 50 mM NaCl),washing said column with low salt solution thereby eluting otherproteins, and eluting the recombinant heparanase from the column by asalt gradient (e.g., 50 mM to 1 M NaCl) or a higher concentration ofsalt (e.g., about 0.4 M).

According to a further aspect of the present invention there is providedan antibody comprising an immunoglobulin elicited against recombinantnative heparanase. The immunoglobulin therefore recognizes and bindsnative (i.e., non denatured) natural or recombinant heparanase.

As used herein in the specification and in the claims section below, theterm “antibody” include serum immunoglobulins, polyclonal antibodies orfragments thereof or monoclonal antibodies or fragments thereof. Theantibodies are preferably elicited against a surface determinant of theparticulate. Monoclonal antibodies or purified fragments of themonoclonal antibodies having at least a portion of an antigen bindingregion, including such as Fv, F(ab1)2, Fab fragments (63), single chainantibodies (U.S. Pat. No. 4,946,778), chimeric or humanized antibodies(64-65) and complementarily determining regions (CDR) may be prepared byconventional procedure. Purification of the serum immunoglobulinsantibodies or fragments can be accomplished by a variety of methodsknown to those of skill including, but not limited to, precipitation byammonium sulfate or sodium sulfate followed by dialysis against saline,ion exchange chromatography, affinity or immunoaffinity chromatographyas well as gel filtration, zone electrophoresis, etc. (see 66).

According to a further aspect of the present invention there is providedan affinity substrate comprising a solid matrix and an immunoglobulinelicited against recombinant native heparanase being immobilizedthereto. Methods of immobilizing immunoglobulins to solid matrices, suchas cellulose, polymeric beads including magnetic beads, are well knownin the art. One such method is described in the Examples section thatfollows. The solid support according to the present invention can bepacked into an affinity column.

According to a further aspect of the present invention there is provideda method of affinity purifying heparanase. The method is effected by (a)loading a heparanase preparation on an affinity column including a solidmatrix and an immunoglobulin elicited against recombinant nativeheparanase being immobilized thereto; (b) washing the affinity column,e.g., using low, say 0-500 mM, salt solution; and (c) eluting heparanasemolecules being adsorbed on the affinity column via the immunoglobulin,e.g., using a high, say 0.5-1.5 M, salt solution.

According to a further aspect of the present invention there is provideda method of activating a heparanase enzyme comprising the step ofdigesting the heparanase enzyme by a protease. The heparanase enzymeaccording to this aspect of the present invention can be natural orrecombinant, purified, partially purified or non-purified. The proteasecan be of any type, including, but not limited to, a cysteine protease,an aspartyl protease, a serine protease and a meatlloproteinase.Examples of specific proteases associated with the above listed proteasefamilies are provided in the Background section. The use of otherproteases for which heparanase includes a recognition and cleavagesequence is envisaged. According to a preferred embodiment digesting theheparanase enzyme by the protease is effected at a pH in which theprotease is active, preferably most active. It is known that someproteases are most active in acidic pH values whereas other proteasesare most active in basic pH values. The pH value at which a specificprotease is most active can be readily determined by one ordinarilyskilled in the art.

According to a further aspect of the present invention there is provideda method of in vivo inhibition of proteolytic processing of heparanase.The method according to this aspect of the present invention is effectedby in vivo administering a protease inhibitor. The protease inhibitorcan be, for example, a cysteine protease inhibitor, an aspartyl proteaseinhibitor, a serine protease inhibitor or a meatlloproteinase inhibitor.Examples of suitable inhibitors are provided in the Examples sectionthat follows. Some protease inhibitors are used pharmaceutically fortreatment of various conditions. In vivo inhibition of proteolyticprocessing of heparanase by a protease inhibitor can be used fortreatment of cancer, metastatic cancers in particular, in whichheparanase activity is involved, because, as further exemplified in theExamples section that follows, the preheparanase (non-processed, p70heparanase) is characterized by lower activity as compared to itsprocessed counterpart (p52 heparanase).

According to a further aspect of the present invention there is provideda nucleic acid construct comprising a first nucleic acid segmentencoding for an upstream (N terminal) portion of heparanase, a second,in frame, nucleic acid sequence encoding a recognition and cleavagesequence of a protease and a third, in frame, nucleic acid sequenceencoding for a downstream portion (C terminal) of heparanase, whereinthe second nucleic acid sequence is in between the first nucleic acidsequence and the third nucleic acid sequence. Examples of suchconstructs are provided in the Examples section that follows.Preferably, the protease is selected having no recognition and cleavagesequences in the upstream and the downstream portions of heparanase,such that when expressed the modified heparanase is digested only at theintroduced recognition and cleavage sequence of the protease.Preferably, the third nucleic acid sequence encodes for a catalyticallyactive heparanase when correctly folded. However, embodiments whereinthe second nucleic acid sequence is so positioned such that whenexpressed the modified heparanase protein is digestible into portionslacking catalytic activity are also envisaged. Such embodiments can beused to provide a heparanase species having a shorter half life, in, forexample, physiological conditions, as compared with the non-modifiedenzyme. One ordinarily skilled in the art would know how to selectlocations for introduction of the recognition and cleavage sequence suchthat the sequence will not hamper the catalytic activity of the enzymeprior to cleavage thereof by the protease.

The above construct, when introduced into a cell expression system canbe used to provide a precursor heparanase protein comprising an upstreamportion of heparanase, a mid portion of a recognition and cleavagesequence of a protease and a downstream portion of heparanase, whereinthe protease is selected having no recognition and cleavage sequences inthe upstream and the downstream portions of heparanase. The recognitionand cleavage sequence of the protease is composed either entirely fromamino acids which are not present in natural heparanase, or from aminoacids which are not present in natural heparanase in part, and furtherfrom adjacent amino acids which are present in natural heparanase.Further according to the present invention there is provided aheparanase protein resulting by digesting the precursor heparanaseprotein described herein.

According to a further aspect of the present invention there is provideda method of obtaining a homogeneously processed, active heparanase. Themethod according to this aspect of the present invention is effected by(a) expressing the precursor heparanase protein in a cell which secretesthe precursor heparanase protein into the growth medium to obtain aconditioned growth medium, the precursor heparanase protein including anupstream portion of heparanase, a mid portion of a recognition andcleavage sequence of a protease and a downstream portion of heparanase,wherein the protease is selected having no recognition and cleavagesequences in the upstream and the downstream portions of heparanase; (b)treating the precursor heparanase protein with the protease; and (c)purifying a proteolytic heparanase product having heparanase catalyticactivity.

It will be appreciated that the various heparanase species describedherein, either activated and/or precursors can be used to producepharmaceutical compositions, including, in addition to heparanase, apharmaceutically acceptable carrier. Affinity purified and proteasetreated, modified, recombinant heparanase is of particular interest forpharmaceutical applications due to its homogeneity and purity.

The present invention has advantages because it provides means forexpressing, purifying and activating recombinant/natural heparanase.Such heparanase can be used in pharmaceutical compositions (see, forexample, U.S. patent application Ser. No. 09/046,465, in whichheparanase is used in the treatment of CF), or as a source of enzyme forhigh throughput heparanase activity assay, which can be used forefficient screening of specific heparanase inhibitors (see, for example,U.S. patent application Ser. No. 09/113,168). By identifying theheparanase proteolytic activation process, novel indirect methods of invivo heparanase inhibition by administration of protease inhibitors wereconceived and tested in vitro. By identifying the heparanase proteolyticactivation process, novel constructs encoding novel heparanase specieshas been constructed and can be used to direct the expression of aheparanase which is homogeneously processed and activated oralternatively neutralized by a dedicated protease.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures inrecombinant DNA technology described below are those well known andcommonly employed in the art. Standard techniques are used for cloning,DNA and RNA isolation, amplification and purification. Generallyenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturers'specifications. Similarly, standard techniques are used for theproteolysis of heparanase by various proteases. These techniques andvarious other techniques used while reducing the present invention topractice are generally performed according to Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989), which is incorporated herein be reference. Othergeneral references are provided throughout this document. The procedurestherein are believed to be well known in the art and are provided forthe convenience of the reader. All the information contained therein isincorporated herein by reference.

Example 1 Expression of Recombinant Human Heparanase in BacteriaExperimental Methods

Construction of Expression Vector

A 1.6 kb fragment of hpa cDNA (SEQ ID NO:1) was amplified from pfasthpa(hpa cDNA cloned in pFastBac, see U.S. patent application Ser. No.08/922,170) by PCR using specific sense primer:Hpu-550bNde—5′-CGCATATGCAGGACGTCGTGGACCTG-3′ (SEQ ID NO:4) and a vectorspecific antisense primer: 3′pFast 5′-TATGATCCTCTAGTACTTCTCGAC-3′ (SEQID NO:5). PCR conditions were: denaturation—94° C., 40 seconds, firstcycle 3 minutes; annealing—58° C., 60 seconds; and elongation—72° C.,2.5 minutes, total of 5 cycles, and then denaturation—94° C., 40seconds; annealing—68° C., 60 seconds; and elongation—72° C., 2.5minutes, for additional 25 cycles.

The Hpu-550Nde primer introduced an NdeI site and an in frame ATG codonpreceding nucleotide 168 of hpa. The PCR product was digested by NdeIand BamHI and its sequence was confirmed with vector specific and genespecific primers, using an automated DNA sequencer (Applied Biosystems,model 373A).

A 1.3 kb BamHI-KpnI fragment was cut out of pFasthpa. The two fragmentswere ligated with the pRSET bacterial expression vector (Invitrogen,CA.).

The resulting plasmid, designated pRSEThpaS1, encoded an open readingframe of 508 amino acids (36-543, SEQ ID NO:2) of the heparanaseprotein, lacking the N-terminal 35 amino acids which are predicted to bea signal peptide.

Transformation: Transformation of E. coli BL21(DE3)pLysS cells(Stratagene) was performed following Stratagene's protocol. Briefly,using β-mereaptoethanol in the transformation buffer cells weretransformed by five seconds of heat shock at 42° C.

Expression of recombinant heparanase: E. coli BL21(DE3)pLysS cellstransformed with the recombinant plasmid were grown at 37° C. overnightin Luria broth (LB) medium containing 100 μg/ml ampicillin and 34 μg/mlchloramphenicol. Cells were diluted {fraction (1/10)} in the samemedium, and the cultures were grown to an OD600 of approximately 0.5.Isopropyl-thiogalactoside (IPTG) (Promega) was added to a finalconcentration of 1 mM and the culture was incubated at 37° C. for 3hours. Cells from IPTG induced cultures were cooled on ice andsedimented by centrifugation at 4,000×g for 20 minutes at 4° C., andresuspended in 0.5 ml of cold phosphate-buffered saline (PBS). Cellswere lysed by sonication, and cell debris were sedimented bycentrifugation at 10,000×g for 20 minutes. The resulting pellet wasanalyzed for proteins by 10% SDS-PAGE, essentially as described inHarlow, E. and Lane, D. Eds. in Antibodies, a laboratory manual. CSHLaboratory press. New-York.

Experimental Results

The expression of recombinant heparanase in E. coli BL21(DE3)pLysS cellscontaining the pRSEThpaS1 was analyzed by SDS-PAGE followed by commassieblue staining for proteins. Bacterial cells were fractionated and aprotein of approximately 70 kDa, which is the expected size of therecombinant heparanase, was observed in the insoluble fraction (FIG. 1,lanes 2-3). That band did not appear when negative control cellstransformed with pRSET were employed (FIG. 1, lane 1).

The identification of the recombinant heparanase expressed in E. coliwas confirmed by a Western blot (data not shown) using a rabbitanti-heparanase polyclonal antibody (disclosed in U.S. patentapplication Ser. No. 09/071,739), followed by ECL detection (Amersham,UK).

As compared to known quantities of co-size separated and stained BSA,the estimated yield of the heparanase recombinant protein under theconditions described was about 0.2 mg/ml of culture (not shown). Theprotein was found in the insoluble fraction (inclusion bodies) and hadno enzymatic activity, as was determined by the soluble ³⁵S-ECMdegradation assay (not shown), however, the recombinant heparanaseprotein expressed in E. coli could provide a source for large quantitiesof heparanase.

It will be appreciated that solubillization and refolding of recombinantproteins expressed in E. coli are well known in the art (see, forexample, for insulin, 70; others are reviewed in 71) and theseprocedures should be applied in order to obtain a functional proteinhaving heparanase activity.

The expression of the recombinant heparanase in bacterial cells is thusdemonstrated in this Example. It will be further appreciated thatchanges in protein length and/or amino acid composition might affect theefficiency of expression, correct folding and the potential yield offunctional enzyme.

Example 2 Expression of Recombinant Human Heparanase in YeastExperimental Methods

Construction of Expression Vectors for Expression in Yeast

Two expression vectors were constructed for the expression of hpa inPichia pastoris. The first vector, designated pPIC3.5K-Sheparanase (FIG.2) contains nucleotides 63-1694 of the hpa sequence (SEQ ID NO:1) clonedinto the expression vector pPIC3.5K (Invitrogen, CA) using a multistepprocedure as follows.

A pair of primers: HPU-664I—5′-AGGAATTCACCATGCTGCTGCGCTCGAAGCCTGCG-3′(SEQ ID NO:6) and HPL-209 5′-GAGTAGCAATTGCTCCTGGTAG-3′ (SEQ ID NO:7)were used in PCR amplification to introduce an EcoRI site just upstreamto the predicted methionine. PCR conditions were: denaturation—94° C.,40 seconds; annealing—50° C., 80 seconds; and elongation—72° C., 180seconds, total of 30 cycles.

The resulting PCR product was digested with EcoRI and BamHI and clonedinto the EcoRI-BamHI sites of the vector phpa2 (described in U.S. patentapplication Ser. No. 08/922,170). The hpa coding region was then removedas an EcoRI-NotI fragment and cloned into the EcoRI-NotI sites of theexpression vector pPIC3.5K to generate the vector pPIC3.5K-Sheparanase(FIG. 2).

The second vector, designated pPIC9K-PP2 (FIG. 3), includes the hpacoding region except for the predicted signal sequence (N-terminal 36amino acids, see SEQ ID NO:2). The hpa was cloned in-frame to the −factor prepro secretion signal in the Pichia pastoris expression vectorpPIC9K (Invitrogen, CA). A pair of primers: HPU-559S,5′-GTCTCGAGAAAAGACAGGACGTCGTGGACCTGGAC-3′ (SEQ ID NO:8) and HPL-209 (SEQID NO:7, described above) were used in PCR amplification under theconditions described above.

The resulting PCR product was digested with XhoI and BamHI and insertedinto the XhoI-BamHII sites of the vector phpa2 (U.S. patent applicationSer. No. 08/922,170).

Thereafter, the XhoI-NotI fragment containing the hpa sequence wasremoved and cloned into an intermediate vector harboring the SacI-NotIsites of pPIC9K.

The hpa was removed from the later vector as a SacI-NotI fragment andcloned into the SacI-NotI sites of pPIC9K, thus creating the vectorpPIC9K-PP2 (FIG. 3).

Transformation and screening: Pichia pastoris strain SMD1168 (his3,pep4) (Invitrogen, CA) was used as a host for transformation.Transformation and selection were carried out as described in the Pichiaexpression Kit protocol (Invitrogen, CA). In all transformations theexpression vectors were linearized with SalI prior to their introductioninto the yeast cells.

Multiple copies integration clones were selected using G-418 (BoehringerMannheim, Germany). Following transformation the top agar layercontaining the yeast cells was removed and re-suspended in 10ml ofsterile water. Aliquots were removed and plated on YPD plates (1% yeastextract, 2% peptone, 2% glucose) containing increasing concentrations ofG-418 (up to 4 mg/ml). Single isolates were picked and streaked on YPDplates. G-418 resistance was then further confirmed by streakingisolates on YPD-G-418 plates.

Expression Experiments

Single colonies were inoculated into 6 ml BMGY—Buffered Glycerol-complexMedium (1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0,1.34% yeast nitrogen base with ammonium sulfate without amino acids,4×10⁻⁵ biotin and 1% glycerol) and incubated at 30° C. at 250 RPM for 24hours. Cells were harvested using clinical centrifuge and re-suspendedin 2.5 ml of BMMY—Buffered Methanol-complex Medium (The same as BMGYexcept that 0.5% methanol replaces the 1% glycerol). Cells were thenincubated at 30° C. at 250 RPM agitation for 48 hour. Culturesupernatants were separated on SDS-PAGE and electrophoreticallytransferred to a nitrocellulose membrane using the Hoeffer-Pharmaciaapparatus, according to manufacturer protocol. A rabbit anti-heparanasepolyclonal antibody (disclosed in U.S. patent application Ser. No.09/071,739) was used as a primary antibody in detection of heparanase.Horseradish peroxidase-labeled anti-rabbit antibodies and ECL Westernblotting detection reagents (Amersham, UK) were used in subsequentdetection steps.

Experimental Results

Both pPIC3.5K-Sheparanase and pPIC9K-PP2 Pichia pastoris transformantssecreted a protein with a similar molecular weight of about 70 kDa, asexpected for heparanase. These results indicate that the heparanasecontains a signal sequence which efficiently functions as a secretionsignal in Pichia pastoris.

G-418 resistance was used to select isolates characterized by multiplegene integration events. A faint heparanase band was observed in thesupernatant of pPIC3.5K-Sheparanase transformant isolated withoutselection on G-418 (FIG. 4, lane 1), whereas no band was observed in thecorresponding position in pPIC3.5K transformant, which served asnegative control (FIG. 4, lane 2). A profound increase in the secretionof heparanase was observed in isolates resistance to 4 mg/ml of G-418(FIG. 4, lanes 3-6).

Example 3 Expression and Secretion of Recombinant Human Heparanase inMammalian Cells Experimental Methods

Construction of hpa DNA Expression Vectors

A hpa gene fragment was cloned under the control of either SV40 earlypromoter (pShpa, FIG. 20a) or the CMV promoter (pChpa, FIG. 20e). Oneconstruct (pShpaCdhft, FIG. 20b) also includes a selection marker, themouse dhfr gene.

Specifically, a 1740 bp hpa gene fragment encoding for a 543 amino acidprotein was introduced into pSI (Promega, USA) or pSI-Cdhfr vectors toyield vectors pShpa and pShpaCdhfr, respectively (FIGS. 5a and 5 b and20 a and 20 b). In both cases the gene was inserted under the SV40 earlypromoter regulation. pShpaCdhfr also carries an expression unit of mousedhfr gene under the regulation of CMV promoter. Another plasmid, pCdhfr(FIG. 20f), included expression unit of mouse dhfr gene under theregulation of CMV promoter and served as control.

A vector designed pS1hpa (FIG. 5c, 20 c) was constructed by ligating atruncated hpa gene fragment (nucleotides 169-1721 of SEQ ID NO:1) to aheterologous signal peptide as follows. Preprotrypsin (PPT) signalpeptide (72) was generated by chemically synthesizing two complementaryoligonucleotides corresponding to the signal peptide encoding DNAsequence, the first having a sequence5′-AATTCACCATGTCTGCACTTCTGATCCTAGCTCTTGTTGGAGCTGCAGTTGCTCAGGAC-3′ (SEQID NO:9), whereas the second having a complementary sequence5′-CCTGAGCAACTGCAGCTCCAACAAGAGCTAGGATCAGAAGTGCAGACATGGTG-3′ (SEQ IDNO:10). Annealing of the complementary oligonucleotides produced thedouble strand sequence encoding to the PPT signal peptide flanked by asticky end of an EcoRI restriction site on the 5′ end thereof and asticky end of an AatII restriction site on the 3′ end thereof. Followingrestriction by EcoRI and AatII of the pfasthpa vector, a 145 bp fragmentwas removed, and replaced by the 52 bp PPT DNA sequence to yield plasmidpS1hpa. The insert thereof was cut out with EcoRI and DotI and ligatedinto the vector pSI.

A vector designed pS2hpa (FIG. 5d and 20 d) was constructed by ligatinga truncated hpa gene fragment (nucleotides 144-1721) to the PPT signalpeptide as follows. Preprotrypsin (PPT) signal peptide (72) wasgenerated by chemically synthesizing two complementary oligonucleotidescorresponding to the signal peptide encoding DNA sequence, the firsthaving a sequence5′-AATTCACCATGTCTGCACTTCTGATCCTAGCTCTTGTTGGAGCTGAGTTGC-3′ (SEQ IDNO:11), whereas the second having a complementary sequence5′-CGGCAACTGCAGCTCCAACAAGAGCTAGGATCAGAAGTGCAGACATGGTG-3′ (SEQ ID NO:12).Annealing of the complementary oligonucleotides produced the doublestrand sequence encoding to the PPT signal peptide flanked by a stickend of an EcoRI restriction site on the 5′ end thereof and a sticky endof a NarI restriction site on the 3′ end thereof.

Following restriction by EcoRi and NarI of pS1hpa plasmid, a 112 bpfragment was removed therefrom and replaced by the PPT DNA sequence togive plasmid pS2hpa (FIG. 5d, 20 d).

Transfection of Vectors into Cells

DNA constructs were introduced into animal cells using thecalcium-phosphate co-percipitaion technique essentially as described in(73).

Selection for dhfr Expressing Stable Cellular Clones

Following transfection, cells were incubated for 48 hours in anon-selective growth medium (F12 medium supplemented with 10% fetal calfserum). Then, the medium was changed to a selection medium (DMEMsupplemented with 10% dialyzed calf serum) and cells were propagated toconfluence at 37° C., under 8% CO₂ aeration. Methotrexate (MIX, 5000 nM)was added to the growth selection medium and resistant cellular cloneswere isolated. Alternatively, cells were transferred after transfectiondirectly to a selection medium containing MTX (100-1000 nM).

SDS Polyacrylamide Gel Electrophoresis and Western Blot Analysis

Denatured and reduced samples were loaded on ready made gradient (4-20%)gels (Novex, USA) and separated under standard gel running conditions(as described in Protein Electrophoresis Application Guide, Hoeffer,U.S.A.). Transfer of proteins onto a PVDF membrane was performedelectrophoretically by a protein transfer apparatus (Hoeffer-Pharmacia).Detection of specific protein was accomplished by a rabbitanti-heparanase polyclonal antibody (disclosed in U.S. patentapplication Ser. No. 09/071,739) (×2000 dilution), followed by ECLdetection (Amersham, UK).

Determination of Heparanase Activity

ECM-derived soluble HSPG assay was performed by incubating cell extractswith solubillized ³⁵S-labeled ECM (18 hours, 37° C.) in the presence of20 mM phosphate buffer (pH 6.2), and size fractionation of thehydrolyzed fraction of the ECM by gel filtration on a Sepharose CL-6Bcolumn. Radiolabeling of degradation fragments eluted at 0.5<Kav<0.8(peak II) was determined (61).

Alternatively, degradation of soluble high molecular weight heparansulfate or heparin molecules to smaller fragments was detected bypolyacrylamide gel electrophoresis analysis. Polyacrylamide gels (7.5%)were loaded with 2.5 mg heparin that was either untreated or incubatedwith heparanase containing cell extracts or media. Staining by methylenblue (74) enabled detection of the heparin molecules and its degradationproducts. The mobility of the molecules on the gel reflects their size.Therefore, activity of heparanase is reflected in a larger quantity ofrapidly migrating heparin degradation products.

Induction of Secretion

CHO stable clones and untransfected CHO cells were induced for secretionof proteins by either calcium ionophore calcimycin (A23187) (Sigma) orphorbol 12-myristate 13-acetate (PMA, Sigma), at differentconcentrations (0.01, 0.1 and 1.0 mg/ml), for various incubation times(2, 8, 24, 48 hours). Induction was performed in the absence of serum.Conditioned medium was collected with 10% buffer citrate pH 5.6 and 200KIU/ml aprotinin (Protosol, Rad Chemicals, Israel), centrifuged toremove floating cells, and kept at −200° C. The amount of secretedprotein(s) was detected by Western blot analysis, and its activity wasdetermined by ³⁵S-ECM degradation assay and soluble heparan sulfatesubstrate hydrolysis assay. When necessary conditioned medium wasconcentrated by ultrafiltration through a 10 kDa filter (Millipore).

Large Scale Propagation of Animal Cells in a Spinner-Basket Bioreactor

The structure and mode of operation of the bioreactor is described indetail in reference 75. A Spinner Basket bioreactor (500 ml, NewBrunswick Scientific) embedded with 10 grams of Fibracel discs(Sterillin, U.K.) was inoculated with seeding inoculum of 1.5×10⁸ cellsof a stable clone of CHO cells designated GGG₁₁ that constitutivelyproduces recombinant heparanase. Propagation of cells was performed in amedium containing 10% serum and cell proliferation was monitored bymeasurement of glucose consumption.

Then growth medium was replaced with medium without serum, suitable forthe production of the recombinant protein. This medium served as asource for recombinant heparanase for later purification.

Experimental Results

Expression of hpa DNA in Animal Cells

Expression of recombinant hpa gene products was detected in a humankidney fibroblasts cell line (293), baby hamster kidney cells (BHK21)and Chinese hamster ovary (CHO dhfr-) cells, following transfection withthe hpa gene (FIGS. 6a-b).

Analysis of recombinant heparanase by Western blotting revealed twodistinct specific protein products: a large protein of about 70 kDa anda predominant protein of about 52 kDa (FIGS. 6a-b).

Transient expression of heparanase proteins was monitored 24-72 hourspost transfection in various cell types.

Human fibroblasts (293 cell line) transfected with pShpa (FIG. 5a) orpChpa constructs (FIG. 5e) exhibited heparanase activity (FIG. 6a, lane4, Table 1 below).

Transfection of CHO cells with the expression vector pShpaCdhfr (FIG.5b) and subsequent selection for MTX resistant clones resulted in theisolation of numerous clones. These cellular clones express hpa geneproducts in a constitutive and stable manner (FIG. 6a, lanes 1-3).

Several CHO cellular clones have been particularly productive inexpressing hpa proteins, as determined by protein blot analysis and byactivity assays (FIG. 6a, FIG. 6b, lane 1, and Table 1). Although thehpa DNA encodes for a large 543 amino acids protein (expected molecularweight about 70 kDa) the results clearly demonstrate the existence oftwo proteins, one of about 70 kDa and another of about 52 kDa. Theseobservations are similar to the results of the transient hpa geneexpression in human 293 cells (FIG. 6a, lane 4). Transient expression ofpShpaCdhfr in CHO cells revealed predominantly a 52 kDa heparanaseprotein (FIG. 6b, lane 2).

It has been previously shown that a 52 kDa protein with heparanaseactivity was isolated from placenta (61) and from platelets, (62). It isthus likely that the 70 kDa protein is naturally processed in the hostcell to yield the 52 kDa protein.

Heparanase Secretion into the Growth Medium

For large scale production and purification purposes, secretion of therecombinant protein into the growth medium is highly desirable.Therefore, expression vectors were constructed (pS1hpa and pS2hpa, FIGS.5c-d) that would drive translation of heparanase attached to the PPTsignal peptide.

Both pS1hpa and pS2hpa plasmids directed the expression of proteinproduct with heparanase activity in human 293 or CHO cells (Table 1).The heparanase was not secreted to the medium in CHO cells. However,transient expression of heparanase encoded by pS1hpa and pS2hpa in human293 cells resulted in the appearance of a single size (about 65 kDa)heparanase protein (FIG. 7c, lanes 3-6).

TABLE 1 Determination of Heparanase activity in transfected animal cellstransfected Heparanase Cell type DNA Activity Human 293 pChpa +(a) cellsHuman 293 pShpa +(b) cells Human 293 pS1hpa +(b) cells Human 293 pS2hpa+(b) cells CHO pShpaCdhfr +(a) Cell extracts were assayed for heparaneseactivity using ECM-derived soluble HSPG assay (a) or direct hydrolysisof soluble substrate (b). Activity detected either in transientlyexpressing cells (293, CHO) or stable cellular clones (CHO).

In order to induce secretion of the recombinant protein(s) into themedium, stable clones and untransfected CHO cells were induced witheither calcium ionophore or PMA. The results show that induction with 1mg/ml calcium ionophore for 2 hours stimulates the secretion of proteinof about 52 kDa from stable clones but not from untransfected cells(data not shown) or untreated stable clones, while longer (24-48 hours)incubation time with 100 ng/ml of calcium ionophore inducespredominantly the secretion of protein of about 70 kDa from stableclones (FIGS. 7a-b). The conditioned medium obtained from the treatedstable clone, which exhibited the 52 kDa protein, had strong heparanaseactivity in ECM-derived soluble HSPG assay (FIGS. 8b-c), and inconcentrated conditioned medium, in the gel shift assay (FIG. 8a). Theheparanase activity in the conditioned medium from the treated stableclone, which exhibited the 70 kDa, is lower than that of the 52 kDafraction (FIGS. 8d-g), since it was active when diluted eight fold whilethe 70 kDa protein failed to show activity in this dilution. It is thuspossible that the 52 kDa protein is the active form of a less active preheparanase of 70 kDa, which is naturally processed to yield themature-active 52 kDa heparanase.

Large Scale Production of Heparanase

Large scale propagation of heparanase expressing cells was set up in a500 ml volume Spinner-Basket bioreactor to demonstrate the ability toobtain a dense adherent cell culture for large scale production ofheparanase. Heparanase constitutively producing cell line was propagatedin the Spinner-Basket bioreactor and at the end of the proliferationphase the medium was replaced with production medium which has the samecomposition as the growth medium but without serum. Cell proliferationand viability were constantly monitored by daily measurements of glucoseconcentration in the medium. Level of glucose was also the parameterused to determine the frequency of medium refreshment in the bioreactor,as described in reference 76. Results of a typical “batch run” thatincludes proliferation and maintenance of heparanase producing cells ina 500 ml Spinner-Basket are shown in FIG. 9.

A “batch run” in a Spinner-Basket bioreactor can last about four weeks,when serum is omitted from the culture medium. The apparatus can belinearly enlarged to bioreactors of 5, 7 or 35 liters. Accordingly,larger amounts of Fibracel can be packed in those vessels andaccommodate, proportionally, larger numbers of cells. The bioreactorscan support cell growth for weeks, or even months, depending on thenature of the cell line and the composition of medium.

Example 4 Expression of Recombinant Heparanase in Virus Infected InsectCells Experimental Methods

Cells

High five and Sf21 insect cell lines were maintained as monolayercultures in SF900II-SFM medium (GibcoBRL).

Recombinant Baculovirus

Recombinant virus containing the hpa gene was constructed using the Bacto Bac system (GibcoBRL). The transfer vector pFastBac (see U.S. patentapplication Ser. No. 08/922,180) was digested with SalI and NotI andligated with a 1.7 kb fragment of phpa2 digested with XhoI and NotI. Theresulting plasmid was designated pFasthpa2. An identical plasmiddesignated pFasthpa4 was prepared as a duplicate and both independentlyserved for further experimentations. Recombinant bacmid was generatedaccording to the instructions of the manufacturer with pFasthpa2,pFasthpa4 and with pFastBac. The latter served as a negative control.Recombinant bacmid DNAs were transfected into Sf21 insect cells. Fivedays after transfection recombinant viruses were harvested and used toinfect High five insect cells, 3×10⁶ cells in T-25 flasks. Cells wereharvested 2-3 days after infection. 4×10⁶ cells were centrifuged andresuspended in a reaction buffer containing 20 mM phosphate citratebuffer, 50 mM NaCl. Cells underwent three cycles of freeze and thaw andlysates were stored at −80° C. Conditioned medium was stored at 4° C.

Experimental Results

Degradation of Soluble ECM-derived HSPG

Monolayer cultures of High five cells were infected (72 h, 28° C.) withrecombinant bacoluvirus containing the pFasthpa plasmid or with controlvirus containing an insert free plasmid. The cells were harvested andlysed in heparanase reaction buffer by three cycles of freezing andthawing. The cell lysates were then incubated (18 h, 37° C.) withsulfate labeled, ECM-derived HSPG (peak I), followed by gel filtrationanalysis (Sepharose 6B) of the reaction mixture.

As shown in FIG. 10, the substrate alone included almost entirely highmolecular weight (Mr) material eluted next to V_(O) (peak I, fractions5-20, Kav<0.35). A similar elution pattern was obtained when the HSPGsubstrate was incubated with lysates of cells that were infected withcontrol virus. In contrast, incubation of the HSPG substrate withlysates of cells infected with the hpa containing virus resulted in acomplete conversion of the high Mr substrate into low Mr labeleddegradation fragments (peak II, fractions 22-35, 0.5<Kav<0.75).

Fragments eluted in peak II were shown to be degradation products ofheparan sulfate, as they were (i) 5- to 6-fold smaller than intactheparan sulfate side chains (Kav approx. 0.33) released from ECM bytreatment with either alkaline borohydride or papain; and (ii) resistantto further digestion with papain or chondroitinase ABC, and susceptibleto deamination by nitrous acid. Similar results (not shown) wereobtained with Sf21 cells. Again, heparanase activity was detected incells infected with the hpa containing virus (pFhpa), but not withcontrol virus (pF). This result was obtained with two independentlygenerated recombinant viruses. Lysates of control not infected High fivecells failed to degrade the HSPG substrate.

In subsequent experiments, the labeled HSPG substrate was incubated withmedium conditioned by infected High five or Sf21 cells.

As shown in FIGS. 11a-b, heparanase activity, reflected by theconversion of the high Mr peak I substrate into the low Mr peak II whichrepresents HS degradation fragments, was found in the growth medium ofcells infected with the pFhpa2 or pFhpa4 viruses, but not with thecontrol pF1 or pF2 viruses. No heparanase activity was detected in thegrowth medium of control non-infected High five or Sf21 cells.

The medium of cells infected with the pFhpa4 virus was passed through a50 kDa cut off membrane to obtain a crude estimation of the molecularweight of the recombinant heparanase enzyme. As demonstrated in FIG. 12,all the enzymatic activity was retained in the upper compartment andthere was no activity in the flow through (<50 kDa) material. Thisresult is consistent with the expected molecular weight of the hpa geneproduct.

In order to further characterize the hpa product the competition effectof heparin, additional substrate of heparanase was examined.

As demonstrated in FIGS. 13a-b, conversion of the peak I substrate intopeak II HS degradation fragments was completely abolished in thepresence of heparin.

Altogether, these results indicate that the heparanase enzyme isexpressed in an active form by insect cells infected with Baculoviruscontaining the newly identified human hpa gene.

Degradation of HSPG in Intact ECM

Next, the ability of intact infected insect cells to degrade HS inintact, naturally produced ECM was investigated. For this purpose, Highfive or Sf21 cells were seeded on metabolically sulfate labeled ECMfollowed by infection (48 h, 28° C.) with either the pFhpa4 or controlpF2 viruses. The pH of the medium was then adjusted to pH 6.2-6.4 andthe cells further incubated with the labeled ECM for another 48 h at 28°C. or 24 h at 37° C. Sulfate labeled material released into theincubation medium was analyzed by gel filtration on Sepharose 6B.

As shown in FIGS. 14a-b and 15 a-b, incubation of the ECM with cellsinfected with the control pF2 virus resulted in a constant release oflabeled material that consisted almost entirely (>90%) of high Mrfragments (peak I) eluted with or next to V_(o). It was previously shownthat a proteolytic activity residing in the ECM itself and/or expressedby cells is responsible for release of the high Mr material. This nearlyintact HSPG provides a soluble substrate for subsequent degradation byheparanase, as also indicated by the relatively large amount of peak Imaterial accumulating when the heparanase enzyme is inhibited by heparin(FIG. 17). On the other hand, incubation of the labeled ECM with cellsinfected with the pFhpa4 virus resulted in release of 60-70% of theECM-associated radioactivity in the form of low Mr sulfate-labeledfragments (peak II, 0.5<Kav<0.75), regardless of whether the infectedcells were incubated with the ECM at 28° C. or 37° C. Control intactnon-infected Sf21 or High five cells failed to degrade the ECM HS sidechains.

In subsequent experiments, as demonstrated in FIGS. 16a-b, High five andSf21 cells were infected (96 h, 28° C.) with pFhpa4 or control pF1viruses and the growth medium incubated with sulfate-labeled ECM. Low MrHS degradation fragments were released from the ECM only upon incubationwith medium conditioned by pFhpa4 infected cells. As shown in FIG. 17,production of these fragments was abolished in the presence of heparin,due to its competitory nature. No heparanase activity was detected inthe growth medium of control, non-infected cells. These results indicatethat the heparanase enzyme expressed by cells infected with the pFhpa4virus is capable of degrading HS when complexed to other macromolecularconstituents (i.e. fibronectin, laminin, collagen) of a naturallyproduced intact ECM, in a manner similar to that reported for highlymetastatic tumor cells or activated cells of the immune system.

Thus, insect cells of several origins (such as Sf21 from Spodopterafrugiperda and High five from Trichoplusia ni) may be infectedproductively with baculovirus. Insect cells are infected withrecombinant baculovirus in which viral DNA sequences have been replacedwith DNA sequences coding for a protein of interest. The protein ofinterest is expressed during the very late phase of infection. A majoradvantage of the baculovirus expression system is that it can be usedfor expressing large amounts of recombinant protein compared to otherpopular expression systems in eukaryotes (e.g., expression in CHOcells). Another advantage of the system is that insect cells have mostof the protein processing capabilities of higher eukaryotic cells. Thus,proteins produced in the recombinant baculovirus-infected cells canundergo co-and post translational processing yielding proteins which aresimilar to the natural protein.

Scaling up the process of culturing and infecting insect cells withbaculovirus is required for the production of recombinant protein ofchoice, in milligram and up to gram quantities. These quantities may berequired for both research or commercial use. Scaling up the processinvolves a variety of fields, such as medium development, metabolicstudies, protein purification and quantification.

Several problems are inherent to this system and effect the process ofscaling up. Upon infection, insect cells become increasingly fragile andsensitive to the physiochemical environment of the culture. One of theprimary goals of the bioengineer is to oxygenate large scale,high-density culture sufficiently, at low shearing rates. Althoughoxygen uptake rates of insect cells are similar to mammalian cell lines,it was found that after infection oxygen uptake rates doubles. Anoptimization process, aimed for setting-up of bioreactor parameters isrequired, for supplying oxygen to the cells without damaging them.

The spinner Bellco, Cat. 1965-56001 was used for scaling up asdescribed. This is a double-wall type spinner. Temperature wascontrolled by water circulated from a 12 liter water bath (FriedElectric, Model TEP1) equipped with a heater and a thermostat. Thespinner was aerated with both air, using an aquarium pump (Rena 301) andoxygen. An oxygen cylinder (medical grade) was connected to the spinnerthrough a two stage regulator set to a pressure of 2 psi. Both air andoxygen were connected to the spinner through a T-connector equipped withvalves that enabled a control over the flow rates of air and of oxygen.A tubing for delivering air mixed with oxygen was connected to thesparger of the spinner through a 0.2-size filter. The sparger used wasof an open type, releasing air-oxygen mixture through an orifice of 3 mminner diameter. The stirring function was provided by a low-RPM magneticstirrer (LH, type 20, LH fermentation Co.), placed beneath the spinner.

High five and Sf21 cells were used alternatively for large scaleproduction of heparanase. Cell culture was gradually built up to1.2×10¹⁰ cells. Eight shake flasks of 500 ml-size were used forculturing cells to 3×10⁶ cells/ml. Cells were cultured with protein-freemedium (Insect-Xpress, Bio Whittaker). 1.5 liters of the above culturewas used for seeding a 6 liters-size spinner. At the time of seeding,culture was diluted to 3 liters with fresh medium. Air was sparged intothe culture at 0.5 liters/min. Stirring rate was 50 RPM and temperaturewas set to 28° C. Two days after seeding, culture volume was doubledagain, from 3 liters to 6 liters. Cell density was adjusted at that timeto 1×10⁶ cells/ml. At that time pure oxygen was sparged at 1.5liters/min in addition to the sparging of air (at 0.5 liters/minute).

Infection of the culture took place one day after doubling the culturevolume from 3 liters to 6 liters, as described. Cells were counted andinfected with the heparanase-coding recombinant virus pFhpa2 at amultiplicity of infection (MOI) of 0.1 or 1.0. The infected culture wasmaintained for approximately 72 hours under conditions set for 6liters-size culture: Oxygen 1.5 liters/min, air 0.5 liters/min,temperature 28° C., agitation at 50 RPM.

Viability of cells in culture was tested every 4 hours, starting from 62hours after virus infection and on. Viability of cells was determined bystaining cells with Trypan Blue dye. The culture was harvested whenviability reached 70-80%. Cells and cell debris were removed bycentrifigation (IEC B-22M, Rotor Cat. 878, 20 min. at 4° ]C. at 7,000RPM). Supernatants were filtered through 0.2 size cartridge (Millipore,Cat. KV0304HB3). Virus and small-size debris were removed with a 300kDa-size cross-flow cartridge (Millipore, Cat. CDUF006LM). Heparanase asconcentrated from filtrate obtained from the 300 kDa-size cartridge with10 kDa size cross-flow cartridge (Millipore, Cat. SK1P003W4). The finalconcentrated solution had a volume of between 0.5 liters and 1 liters.Heparanase was purified from the concentrated solution on HPLC. Table 2below summarizes the results of two large scale heparanase production byinsect cells experiments.

TABLE 2 Harvest Cell Volume time viability heparanase of post at inBatch Cells MOI culture infection harvest harvest No. used used (L)(hours) (%) (mg/ml) 30 Sf21 0.1 4.5 78 76 0.44 31 Hi-5 0.1 6.0 75 760.16

Example 5 Purification of Recombinant Heparanase Experimental Methodsand Results

Methods and Results

Baculovirus infected insect cells (1 or 5 liter of High five cellsuspension) were harvested by centrifugation. The supernatant was passedthrough 0.2 micron filter (Millipore), then filtered through 300Kcartridge (Millipore). The <300 kDa retentate (about 300 ml) was washedby further filtration with 2 volumes of phosphate buffered saline (PBS).The <300 kDa filtrate was then concentrated by 10K cellulose cartridge(Millipore). The >10 kDa retentate was diluted three fold with 10 mMphosphate buffer pH 6.8 to prepare for applying the crude enzymepreparation onto a Source-S column (Pharmacia).

The diluted >10 kDa retentate was subjected to a Source-S column (2.5×10cm) pre equilibrated with 10 mM phosphate buffer pH 6.8, 50 mM NaCl).Most of the contaminating proteins did not bind to the column whileheparanase bound tightly. Heparanase activity was eluted by a lineargradient of 0.05 M NaCl—1 M NaCl in phosphate buffer pH 6.8 andfractions of 5 ml were collected.

The fractions having the highest activity in degrading sulfate labeledECM were combined. The 0.4 M NaCl fractions were about 90% pure andexhibited the highest activity (FIG. 18, lane 9). A rabbitanti-heparanase polyclonal antibody detected the purified enzyme inWestern blot—ECL analysis (FIG. 19, lane 9).

These results demonstrate a powerful single step purification ofrecombinant heparanase from culture supernatants. Obviously, otherpurification methods, such as affinity purification using, for example,solid support bound heparanase substrates, heparanase inhibitors oranti-heparanase antibodies, size exclusion, hydrophobic interactions,etc. can be additionally employed.

Example 6 Purification of Heparanase and Production of Highly ActiveHeparanase Species by Proteolytic Processing Experimental Methods

Construction of hpa DNA expression vectors, transfection thereof intocells, selection for dhfr expressing stable cellular clones, inductionof secretion and SDS polyacrylamide gel electrophoresis and Western blotanalyses were all performed as described hereinabove under Example 3.

Heparanase Activity Using DMB Assay

For each sample, 100 μl heparin sepharose (50% suspension in 1×bufferA—containing 20 mM Phosphate citrate buffer pH 5.4, 1 mM CaCl₂ and 1 mMNaCl) were incubated in 0.5 ml eppendorf tube for 17 hours with a testedenzyme preparation. At the end of the incubation period, samples werecentrifuged for 2 minutes at 1000 rpm and the supernatants were analyzedfor sulfated polyanions (heparin) using the colorimetricdimethylmethylene blue assay as follows.

Supernatants (100 μl) were transferred to plastic cuvettes and dilutedto 0.5 ml with PBS supplemented with 1% BSA. 1,9-Dimethylmethylene blue(32 mg dissolved in 5 ml ethanol and diluted to 1 liter with formatebuffer) (0.5 ml) was added to each cuvette. Absorbency at 530 nm wasdetermined using a spectrophotometer (Cary 100, Varian). For each samplea control, to which the enzyme was added at the end of the incubationperiod, was included. For further details, see U.S. patent Ser. No.09/113,168, which is incorporated by reference as if fully set forthherein.

Heparanase Activity Using the Tetrazolium Assay

Heparanase activity was determined in reactions containing buffer A and50 μg heparan sulfate in a final volume of 100 μl. Reactions wereperformed in a 96 well microtiterplate at 37° C. for 17 hours. Reactionwere thereafter stopped by the addition of 100 μl tetrazolium bluereagent (0.1% tetrazolium blue in 0.1 M NaOH) to each well. Color wasdeveloped following incubation at 60° C. for 40 minutes. Color intensitywas quantitatively determined at 580 nm using a microtiterplate reader(Dynatech). For each sample a control, to which the enzyme was added atthe end of the incubation period, was included. A glucose standard curveof 1-15 μg glucose was included in each assay. Heparanase activity wascalculated as ΔO.D. of the sample containing the substrate minus the O.Dof the control sample. The result was converted to μg glucoseequivalent. One unit is defined as μg glucose equivalent produced perminute. For further details, see U.S. patent Ser. No. 09/113,168, whichis incorporated by reference as if fully set forth herein.

Production of Rabbit Anti-native Heparanase Polyclonal Antibodies

Rabbits were immunized in three two weeks intervals with 200 mg ofpurified human recombinant heparanase protein produced in baculovirusinfected Sf21 insect cells (see Examples 4-5 above) emulsified with anequal volume of complete Freund's adjuvant. Ten days after the thirdimmunization rabbits were bled and serum was examined for reactivitywith recombinant heparanase. Four weeks after bleeding another boost wasinjected and 10 days later blood was collected.

Purification of Heparanase from Mammalian Cell Extract Using IonExchange Chromatography

2TT1 CHO cells (2×10⁸ cells stably transfected with pShpaCdhfr, FIG.20b) were extracted in 2.5 ml of 10 mM phosphate citrate buffer, pH 5.4.The extract was centrifuged at 2,750×g for 5 minutes and the supernatantwas collected for heparanase enzyme purification using cation exchangechromatography as follows. An HPLC column (mono-S HR 5/5, PharmaciaBiotech) was equilibrated with 20 mM sodium phosphate buffer, pH 6.8,and the supernatant was loaded thereon. Proteins were eluted from thecolumn using a linear gradient of 0 to 1 M sodium chloride in 20 mMsodium phosphate buffer, pH 6.8. The gradient was carried out in 20column volumes at a flow rate of one ml per minute. Elution of proteinswas monitored at 214 nm (FIG. 23a) and fractions of 1 ml each werecollected. An aliquot from each fraction was analyzed for heparanaseactivity using the DMB assay and for immunoreactivity using a mouseanti-heparanase monoclonal antibody (see U.S. patent Ser. No.09/071,739, which is incorporated herein by reference). Most of theheparanase was eluted in fractions 19-20.

Preparation of an Affinity Column with Anti-native Heparanase Antibodies

An affinity column was prepared using the Immunopure Protein G IgGOrientation Kit (Pierce). To this end, 17 mg of the above describedrabbit anti-native heparanase polyclonal antibody, purified on protein Gsepharose, were bound to a column containing 2 ml Immunopure immobilizedprotein G. The antibody was cross linked to the protein G with DMP.Unreacted imidate groups were blocked and the column was equilibratedwith 20 mM phosphate buffer, pH 6.8.

Purification of Heparanase Using the Affinity Column

0.5×10⁸ 2TT1 CHO cells were suspended in 2.5 ml of 20 mM phosphatecitrate buffer, pH 5.4. Cells were frozen in liquid nitrogen andsubsequently thawed at 37° C. Freezing and thawing were repeated twomore times. The extract was then centrifuged for 15 minutes at 4000 gand the resulting supernatant was loaded onto the affinity column andwas incubated, to allow binding of the enzyme to the column, at 4° C.for 17 hours under head-over-tail shaking. Thereafter, unbound proteinswere washed until absorbency at 280 nm reached zero. Proteins wereeluted from the column with 0.1 M glycine HCl buffer, pH 3.5. 900 μlfractions were collected into eppendorf tubes each containing 100 μl of1 M phosphate buffer, pH 8. The presence of heparanase in the elutedfractions was determined by Western blotting following gradient 4-20%SDS-PAGE of 20 μl samples using anti-heparanase monoclonal antibody (seeU.S. patent Ser. No. 09/071,739). Heparanase activity was determined in30 μl samples using the above described DMB assay.

Construction of Heparanase Expression Vectors with a Unique ProteaseCleavage Sequence

Expression vectors for the production of a heparanase protein speciescarrying a unique proteolytic cleavage site were designed andconstructed. Two independent sites, just upstream of amino acids 120 or158 (SEQ ID NO:2), both are peaking on the hydropathy plot, ascalculated by the Kyte-Doolittle method for calculating hydrophilicity,using the Wisconsin University GCG DNA analysis software (FIG. 29a),were selected for insertion of either one of two protease recognitionand cleavage sequences within the hpa cDNA sequence to yield twoheparanase species designated herein as pre-p56′ and pre-p52′, which,following digestion with their respective protease, yield truncatedproteins designated herein p52′ and p56′, respectively. A first sequenceincluded 4 amino acids (Ile-Glu-Gly-Arg↓, SEQ ID NO:13) which constitutea factor Xa recognition and cleavage sequence. An alternative, second,sequence included 5 amino acids (Asp-Asp-Asp-Asp-Lys↓, SEQ ID NO:14)which constitute a enterokinase recognition and cleavage sequence. Thesesequences do not appear in the natural enzyme (SEQ ID NO:2).

To this end, the following PCR primers were constructed:52-Xa—5′-CCATCGATAGAAGGACGAAAAAAGTTCAAGAACAGCACCTAC-3′ (SEQ ID NO:15);52 x-Cla—5′-GGATCGATTGGTAGTGTTCTCGGAGTAG-3′ (SEQ ID NO:16);56-Xa—5′-GGATCGATAGAAGGACGATCTCAAGTCAACCAGGATATT-3′ (SEQ ID NO:17);56-xCla—5′-CCATCGATGCCCAGTAACTTCTCTCTTCAAAG-3′ (SEQ ID NO:18); hpl967—5′-TCAGATGCAAGCAGCAACTTTGGC-3′ (SEQ ID NO:19); hpu685—5′-GAGCAGCCAGGTGAGCCCAAGAT-3′ (SEQ ID NO:20); 52-EK5′-CCATCGATGACGACGACAAGAAAAAGTTCAAGAACAGCACCTAC-3′ (SEQ ID NO:21);52e-Cla—5′-GGATCGATCTGGTAGTGTTCTCGGAGTAG-3′ (SEQ ID NO:22);56-EK—5′-GGATCGATGACGACGACAAGTCTCAAGTCAACCAGGATATTTG-3′ (SEQ ID NO:23);and 56e-Cla—5′-CCATCGATTTGGGAGTAACTTCTCTCTTCAAAG-3′ (SEQ ID NO:24).

The following constructs were prepared (FIG. 29b):

(i) Construction of pre-p52′-Xa hpa in pFast: A first PCR reaction wasperformed with a pFasthpa2 template and with primers 52-Xa and hpl 967.The resulted 1180 bp fragment was digested with ClaI and AflII and a 220bp fragment was isolated. A second PCR reaction was performed with apFasthpa2 template and with primers 52x-Cla and hpu 685. The resulting500 bp fragment was digested with ClaI and AatII and a 370 bp fragmentwas isolated. The ClaI-AflII 220 bp and the ClaI-AatII 370 bp fragmentswere ligated to a 5,900 AatII-AflII fragment of the pFasthpa2 plasmid.

(ii) Construction of pre-p56′-Xa hpa in pFast: A first PCR reaction wasperformed with a pFasthpa2 template and with primers 56-Xa and hpl 967.The resulted 1290 bp fragment was digested with ClaI and AflII and a 340bp fragment was isolated. A second PCR reaction was performed with apFasthpa2 template and with primers 56x-Cla and hpu 685. The resulting380 bp fragment was digested with ClaI and AatII and a 250 bp fragmentwas isolated. The ClaI-AflII 340 bp and the ClaI-AatII 250 bp fragmentswere ligated to a 5,900 AatII-AflII fragment of the pFasthpa2 plasmid.

(iii) Construction of pre-p52′-Enterokinase hpa in pFast: A first PCRreaction was performed with a pFasthpa2 template and with primers 52-EKand hpl 967. The resulted 1180 bp fragment was digested with ClaI andAflII and a 220 bp fragment was isolated. A second PCR reaction wasperformed with a pFasthpa2 template and with primers 52e-Cla and hpu685. The resulting 500 bp fragment was digested with ClaI and AatII anda 370 bp fragment was isolated. The ClaI-AflII 220 bp and the ClaI-AatlI370 bp fragments were ligated to a 5,900 AatII-AflII fragment of thepFasthpa2 plasmid.

(iv) Construction of pre-p56′-Enterokinase hpa in pFast: A first PCRreaction was performed with a pFasthpa2 template and with primers 56-EKand hpl 967. The resulted 1290 bp fragment was digested with ClaI andAflII and a 340 bp fragment was isolated. A second PCR reaction wasperformed with a pFasthpa2 template and with primers 56e-Cla and hpu685. The resulting 380 bp fragment was digested with ClaI and AatII anda 250 bp fragment was isolated. The ClaI-AflII 340 bp and the ClaI-AatII250 bp fragments were ligated to a 5,900 AatII-AflII fragment of thepFasthpa2 plasmid.

Construction of Plasmids for Expression of Heparanase with ProteaseDigestion Sequence

Each one of the four constructs (i to iv) described hereinabove includesan AatII-AflII fragment which includes a factor Xa or enterokinaserecognition and cleavage sequence positioned at one of the describedalternative sites, i.e., upstream amino acids 120 or 158 (SEQ ID NO:2).The hpa constructs described in FIGS. 5a-e and 20 a-e, as well as thepFasthpa constructs, each includes a single AatII site and a singleAflII site within the hpa cDNA sequence, thus enabling the insertion byreplacement of the 220 or 340 AatII-AflII fragments as desired.

Experimental Results

Expression of hpa DNA in Animal Cells

As already shown and discussed under Example 3 above, in order to drivetransient or stable expression of the hpa gene in animal cells, the hpagene was cloned into expression vectors, where transcription isregulated by promoters of viral origin (SV40, CMV) to ensure efficienttranscription (FIGS. 5a-e). All vectors were suitable for transientexpression of hpa in animal cells, but only vectors that include anexpression cassette for the mouse dhfr gene (FIGS. 5b and 20 f, thelatter serves as a negative control) could be subjected to selection bymrthotrexate (MTX). Selection enables the establishment of cell linesthat constitutively produce high levels of recombinant heparanase.

Cell lines of different origins have been transfected and expressedhuman heparanase gene: Transient expression of recombinant heparanasewas detected in a human kidney fibroblasts cell line 293 (FIG. 6a), babyhamster kidney cells (BHK21; FIG. 21a) and Chinese hamster ovary cells(CHO; FIG. 6b). Stable expression of heparanase in CHO cells is shown inFIGS. 6a-b.

Transfection of CHO cells with the expression vector pShpaCdhfr (FIG.5b) or co-transfection with pS1hpa and pCdhfr (FIGS. 5c and 20 f),followed by selection for MTX resistant clones resulted in the isolationof numerous clones. These cellular clones express hpa gene products in aconstitutive and stable manner (FIG. 6a, lanes 1-3).

Analysis of expression of recombinant heparanase in mammalian cellsrevealed two distinct specific protein products: a large protein ofabout 70 kDa (which is referred to herein as p70) and a predominantprotein of about 50 kDa, which is referred to herein as p52 (FIGS. 6a,21 a).

Although the hpa DNA encodes a large 543 amino acids protein (expectedmolecular weight about 61 kDa), the results clearly demonstrate theexistence of two proteins. These observations are similar to the resultsof the transient hpa gene expression in human 293 cells (FIG. 6a, lane4). BHK21 cells, transiently transfected with pS1 hpa (FIG. 5c) expresspredominantly the p52 form of recombinant heparanase (FIG. 21a, lane 1marked by an arrow). Stable CHO clones express predominantly the p52protein (FIG. 6b, lane 2).

The presence of both p70 and p52 heparanase was detected in all cellsthat expressed the hpa gene, although the relative concentrations of theproteins varied between different cell types.

Cells transfected with pS1hpa (FIG. 5c) expressed p52 (FIG. 21a)indicating that the replacement of the putative heparanase signalpeptide by the PPT signal sequence did not affect the expression andprocessing of the protein.

All cell extracts exhibited high heparanase activity following theintroduction of the hpa gene. Human. 293 cells transfected with pShpa(FIG. 5e) exhibited high heparanase activity (FIG. 21b).

It has been previously shown that a 52 kDa protein with heparanaseactivity was isolated from placenta (61) an platelets (62).

It is thus concluded that the p70 protein is a preheparanase that isnaturally processed in the host cell to yield the p52 protein.

Heparanase Secretion into the Growth Medium

For large scale production and purification purposes, secretion of therecombinant protein into the growth medium is highly desirable.Therefore, expression vectors were constructed (pS1hpa and pS2hpa, FIGS.5c-d) to direct translation of heparanase attached to the PPT signalpeptide, a secretion signal peptide.

Both pS1hpa and pS2hpa plasmids directed the expression of proteinproduct with heparanase activity in human 293 or CHO cells (FIGS. 7c, 22a-b). Transient expression of heparanase from pS1hpa and pS2hpa resultedin the appearance of a single size (about 70 kDa) heparanase protein inthe medium (FIG. 7c, lanes 3-6), similar to the larger form ofrecombinant heparanase detected in the cells.

CHO cells, stably transfected with either pShpaCdhfr (2TT1 clones) orpS1hpa (S1PPT clones) were further subcloned to yield stable cloneswhich maintain their genetic and cellular characteristics stability inthe absence of MTX selection. To this end, the limiting dilutionprocedure was employed, in which cells were cloned under non-selectiveconditions and clones exhibiting the above stability were selected forfurther analysis.

2TT1 and S1PPT clones under (clones 2TT1 and S1PPT-p) or after (clones2TT1-2, 2TT1-8, S1PPT-4 and S1PPT-8) selection with high MTX yieldedstable clones exhibiting moderate (clones 2TT1 (FIG. 22b), 2TT1-2,2TT1-8) or high (clones S1PPT-p, S1PPT-4, S1PPT-8 (FIG. 22a))constitutive secretion of heparanase into the growth medium. Thesecreted protein was of about 70 kDa, similar to p70, the largerheparanase form found within the cells (FIGS. 22a-b). Only when a largeamount of p70 protein are found in the medium, a residual amount of thesmaller heparanase form, p52, could be detected (FIG. 22a, lane 4).

In the conditioned medium containing heparanase, some heparanaseactivity could be detected, although not as high as the activitymeasured in the respective cell extracts which, as determinedimmunologically, have comparable heparanase concentrations. Someimprovement in secretion could be detected by calcium ionophoretreatment, but the effect was transient (FIG. 22a, lane 4).

The Purification of Recombinant Heparanase from 2TT1 CHO Cells by IonExchange Chromatography

Clone 2TT1-8 was used for large scale production of heparanase. In thiscell line, the p52 form of heparanase is predominantly expressed withinthe cells. The cells are grown adherent to the tissue culture flasksurface and were harvested when the cell culture reaches confluency.

Purification of a,non-abundant protein from cells is a challenging task,where only an carefully designed and accurately discriminating protocolenables purification. See U.S. Pat. No. 5,362,641 and references 61 and62 describing the purification of heparanase from placenta andplatelets.

Here, a cation exchange chromatography procedure was selected forpurification based on successful use thereof in the purification ofinsect cells produced recombinant heparanase, as described in Example 5hereinabove.

Separation of the total protein content of 2TT1-8 cell extract on amono-S cation exchange column is shown in FIG. 23a. The vast majority ofcellular proteins were eluted from the column prior to the elution ofheparanase (FIG. 23b). It is important to note that the p52 and the p70were co-eluted under these conditions. Furthermore, a tight correlationwas found between the presence of heparanase, as detectedimmunologically (FIG. 23b), and its activity, as measured by the DMB(FIG. 23c) and the tetrazolium (FIG. 23d) activity assays.

Thus, using the above described purification protocol, one obtains ampleamounts of highly active and purified heparanase which is highlysuitable for use in a high throughput screening assay for heparanaseactivity, e.g., in the presence of candidate heparanase inhibitors, forexample, combinatorial inhibitor libraries. Further details relating toa heparanase high throughput assay are provided in U.S. patentapplication Ser. No. 09/113,168, which is incorporated herein byreference.

The Purification of Heparanase by an Anti-heparanse Affinity Column

Partially purified, active recombinant heparanse produced in SF21 insectcells infected with a baculovirus containing the hpa cDNA, was used toimmunize rabbits for the production of polyclonal antibodies against thenative recombinant heparanase protein. This antibody was thereafterpurified and was used to construct a heparanase affinity column.

Cellular extract of CHO 2TT1-8 cells was loaded on the column foraffinity separation. FIGS. 24a-b clearly show that heparanase wasspecifically and efficiently bound to the affinity column. Moreover,high salt elution of the bound heparanase from the column was efficientand the activity of the eluted heparanase (FIG. 24b) was tightlycorrelated with the presence of the recombinant enzyme (FIG. 24a). Thus,using an affinity column as herein described, one can obtain a highlypurified and highly active recombinant or natural heparanase in singlestep purification, which can be used in pharmaceutical applications.Furthermore, combining the Mono-S and affinity columns into a two steppurification procedure, will ensure even better results in terms of bothpurification and yield.

In addition, the tetrazolium assay is based on the detection of freereducing sugar ends. As such it requires heparanase preparations devoidof such reducing ends. Heparanase purified using the above describedaffinity column is devoid of such reducing ends, and is therefore highlyapplicable for the tetrazolium activity assay.

Proteolytic Processing of Heparanase by Protease from Insect Cells

Production of human recombinant heparanase in insect cells (Sf21), viabaculovirus infection, and the subsequent purification of that proteinare described in U.S. patent Ser. Nos. 08/922,170; 09/071,618;09/109,386; and in PCT/US98/17954, all of which are incorporated hereinby reference.

Briefly, conditioned medium of Sf21 cells that were infected withrecombinant baculovirus, secrete heparanase to the medium. Thisheparanase is a glycosylated protein with an apparent molecular weightof 70 kDa. The size of that protein is similar to the p70 produced bymammalian cells, and it possesses limited heparanase activity. Thisheparanase protein is referred to herein as p70-bac heparanase.

Purification of p70-bac heparanase from insect cells conditioned mediuminvolved sequential filtration steps and a cation exchange column(Source-S). Fractions that contain predominantly p70-bac heparanaseprotein are collected. This purification protocol and results aredescribed hereinabove.

The effect of different pH values on the activity and intactness ofp70-bac heparanase was examined in an attempt to establish a pH optimumfor heparanase activity. It was found that exposure of p70-bacheparanase to pH 4.0 for one week at 4° C. resulted in significant(seven fold) increase in activity (FIG. 25b). This activation wasprotease dependent as is evident form the inhibition of activationcaused by a protease inhibitors cocktail (FIG. 25b).

The fate of the p70-bac heparanase following exposure to acidic pH wasuncovered by Western-blot analysis (FIG. 25a). Following exposure to pH4, p70-bac heparanase was converted into a lower molecular weight form,of about 56 kDa, which is referred to herein as p56 (FIG. 25a, lane C).Proteolysis was inhibited in the presence of protease inhibitors (FIG.25a, lane B).

This is the first record demonstrating (i) in vitro proteolyticprocessing of recombinant heparanase, (ii) associated with a significantincrease in heparanase activity.

To further characterize the protease(s) involved in processing andactivation of p70-bac heparanase, a collection of individual proteaseinhibitors was employed (FIGS. 25c-d). The inhibitors antipain, E-64, isleupeptin and chemostatin were most effective in preventing theactivation of p70-bac heparanase at low pH. The effect was due toinhibition of the proteolytic processing of the p70-bac heparanase as isevident from the Western blot analysis of FIG. 25c. Antipain andleupeptin are known to inhibit serine and cysteine proteases, while E-64inhibits only cysteine proteases. These results therefore indicate thata cysteine protease(s) present in the conditioned medium of insect cellsare responsible for the activation of p70-bac heparanase, by processingthe enzyme into a lower and more active p56 molecular weight form.

N-terminal sequencing of gel separated and PVDF transferred p56heparanase revealed the sequence Ser-Gln-Val-Asn-Gln (SEQ ID NO:25),which corresponds to a new heparanase species that starts at Ser 120 ofthe full length enzyme (SEQ ID NO:2).

Proteolytic Processing of Heparanase by Trypsin and Cathepsin L

The activation of p70-bac heparanase by protease(s) from insect cellsconditioned medium could be reproduced by mild digestion with trypsin(FIGS. 26a-b). Trypsin, 1.5 to 500 units per 10 μg p70-bac heparanase,gradually activated the protein, reaching maximal activation offive-fold already at 15 units trypsin (FIG. 26a). Activation of p70-bacheparanase correlated with the expected cleavage of a portion of thep70-bac heparanase into smaller heparanase species, of about 56 kDa(FIG. 26b). Smaller fragments of heparanase were also obtained bytrypsinization (FIG. 26b, lanes 2-3).

Similarly, recombinant heparanase processing and activation occurredwhen mild trypsin digestion was employed on a crude conditioned mediumof CHO cells that secrete mammalian p70 heparanase (FIG. 27). Activationwas dose dependent.

Processing and activation of recombinant CHO produced and secretedheparanase (p70) was also obtained by mild treatment with Cathepsin L,which is a known cysteine protease (FIGS. 28a-b). Processing by thisprotease resulted in several digestion products, of about 56, 34 and 21kDa (FIG. 28b, lane 2).

It is shown herein that proteolytic digestion of recombinant heparanasefrom a variety of sources and by a variety of proteases results in (i)processing of the enzyme into a lower molecular weigh species; and (ii)increased catalytic activity. Processing and activation of heparanase ina similar fashion is anticipated to take place in vivo as well andtherefore in vivo inhibition of proteases can be used to indirectlyinhibit heparanase processing and activation.

Design of Expression Vectors to Express Heparanase Precursor SpeciesAdapted for In vitro Activation by Proteases

The p52 heparanase protein (as characterized in CHO, 293 and BHK21cells, placental and platelets heparanase) and the p56 heparanaseprotein (as characterized after processing of the p70-bac heparanase)are presently the forms of heparanase that exhibit the highest enzymaticactivity. It is shown herein that these heparanase species are theresult of proteolytic cleavages of heparanase. As was determined bysolid phase microsequencing the cleavage site of p70-bac heparanase iseffected between amino acids 119 and 120 (SEQ ID NO:2, see above) withinthe first peak of hydrophilicity (FIG. 29a, peak I). The second peak ofhydrophilicity (FIG. 29a, peak II) is expected to contain the cleavagesite yielding the p52 heparanase species. This is not surprising,considering the fact that these regions, are positioned at the surfaceof the heparanase molecule and are thus susceptible to proteolysis.

FIG. 29c demonstrates the steps undertaken in constructing four basicnucleic acid constructs harboring a unique protease recognition andcleavage sequence of factor Xa—Ile-Glu-Gly-Arg↓—or ofenterokinase—Asp-Asp-Asp-Asp-Lys↓ downstream amino acids 119 or 157.AatII-AflII restriction fragments derived from these four basicconstructs can be used to replace a corresponding region in any of thehpa constructs described herein (FIGS. 5a-e) and for that effect, anyother construct harboring a hpa derived sequence. FIG. 29b shows themodified heparanase species (pre-p56′ and pre-p52′) that contain theseunique protease recognition and cleavage sequences (shaded regions)which enable proteolytic processing by the respective proteases toobtain homogeneously processed and highly active heparanase species(p56′ and p52′, respectively).

The above described constructs are highly suitable for expression ofheparanase in any expression system which is characterized by secretionof the recombinant heparanase to the growth medium. Such a precursorenzyme can be readily and precisely processed into a mature active formof heparanase—p56′ or p52′.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

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25 1721 nucleic acid double linear 1 CTAGAGCTTT CGACTCTCCG CTGCGCGGCAGCTGGCGGGG GGAGCAGCCA GGTGAGCCCA 60 AGATGCTGCT GCGCTCGAAG CCTGCGCTGCCGCCGCCGCT GATGCTGCTG CTCCTGGGGC 120 CGCTGGGTCC CCTCTCCCCT GGCGCCCTGCCCCGACCTGC GCAAGCACAG GACGTCGTGG 180 ACCTGGACTT CTTCACCCAG GAGCCGCTGCACCTGGTGAG CCCCTCGTTC CTGTCCGTCA 240 CCATTGACGC CAACCTGGCC ACGGACCCGCGGTTCCTCAT CCTCCTGGGT TCTCCAAAGC 300 TTCGTACCTT GGCCAGAGGC TTGTCTCCTGCGTACCTGAG GTTTGGTGGC ACCAAGACAG 360 ACTTCCTAAT TTTCGATCCC AAGAAGGAATCAACCTTTGA AGAGAGAAGT TACTGGCAAT 420 CTCAAGTCAA CCAGGATATT TGCAAATATGGATCCATCCC TCCTGATGTG GAGGAGAAGT 480 TACGGTTGGA ATGGCCCTAC CAGGAGCAATTGCTACTCCG AGAACACTAC CAGAAAAAGT 540 TCAAGAACAG CACCTACTCA AGAAGCTCTGTAGATGTGCT ATACACTTTT GCAAACTGCT 600 CAGGACTGGA CTTGATCTTT GGCCTAAATGCGTTATTAAG AACAGCAGAT TTGCAGTGGA 660 ACAGTTCTAA TGCTCAGTTG CTCCTGGACTACTGCTCTTC CAAGGGGTAT AACATTTCTT 720 GGGAACTAGG CAATGAACCT AACAGTTTCCTTAAGAAGGC TGATATTTTC ATCAATGGGT 780 CGCAGTTAGG AGAAGATTAT ATTCAATTGCATAAACTTCT AAGAAAGTCC ACCTTCAAAA 840 ATGCAAAACT CTATGGTCCT GATGTTGGTCAGCCTCGAAG AAAGACGGCT AAGATGCTGA 900 AGAGCTTCCT GAAGGCTGGT GGAGAAGTGATTGATTCAGT TACATGGCAT CACTACTATT 960 TGAATGGACG GACTGCTACC AGGGAAGATTTTCTAAACCC TGATGTATTG GACATTTTTA 1020 TTTCATCTGT GCAAAAAGTT TTCCAGGTGGTTGAGAGCAC CAGGCCTGGC AAGAAGGTCT 1080 GGTTAGGAGA AACAAGCTCT GCATATGGAGGCGGAGCGCC CTTGCTATCC GACACCTTTG 1140 CAGCTGGCTT TATGTGGCTG GATAAATTGGGCCTGTCAGC CCGAATGGGA ATAGAAGTGG 1200 TGATGAGGCA AGTATTCTTT GGAGCAGGAAACTACCATTT AGTGGATGAA AACTTCGATC 1260 CTTTACCTGA TTATTGGCTA TCTCTTCTGTTCAAGAAATT GGTGGGCACC AAGGTGTTAA 1320 TGGCAAGCGT GCAAGGTTCA AAGAGAAGGAAGCTTCGAGT ATACCTTCAT TGCACAAACA 1380 CTGACAATCC AAGGTATAAA GAAGGAGATTTAACTCTGTA TGCCATAAAC CTCCATAAGG 1440 TCACCAAGTA CTTGCGGTTA CCCTATCCTTTTTCTAACAA GCAAGTGGAT AAATACCTTC 1500 TAAGACCTTT GGGACCTCAT GGATTACTTTCCAAATCTGT CCAACTCAAT GGTCTAACTC 1560 TAAAGATGGT GGATGATCAA ACCTTGCCACCTTTAATGGA AAAACCTCTC CGGCCAGGAA 1620 GTTCACTGGG CTTGCCAGCT TTCTCATATAGTTTTTTTGT GATAAGAAAT GCCAAAGTTG 1680 CTGCTTGCAT CTGAAAATAA AATATACTAGTCCTGACACT G 1721 543 amino acid single linear 2 Met Leu Leu Arg Ser LysPro Ala Leu Pro Pro Pro Leu Met Leu Leu 5 10 15 Leu Leu Gly Pro Leu GlyPro Leu Ser Pro Gly Ala Leu Pro Arg Pro 20 25 30 Ala Gln Ala Gln Asp ValVal Asp Leu Asp Phe Phe Thr Gln Glu Pro 35 40 45 Leu His Leu Val Ser ProSer Phe Leu Ser Val Thr Ile Asp Ala Asn 50 55 60 Leu Ala Thr Asp Pro ArgPhe Leu Ile Leu Leu Gly Ser Pro Lys Leu 65 70 75 80 Arg Thr Leu Ala ArgGly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly 85 90 95 Thr Lys Thr Asp PheLeu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe 100 105 110 Glu Glu Arg SerTyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys 115 120 125 Tyr Gly SerIle Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp 130 135 140 Pro TyrGln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe 145 150 155 160Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe 165 170175 Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu 180185 190 Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu195 200 205 Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu GlyAsn 210 215 220 Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile AsnGly Ser 225 230 235 240 Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys LeuLeu Arg Lys Ser 245 250 255 Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro AspVal Gly Gln Pro Arg 260 265 270 Arg Lys Thr Ala Lys Met Leu Lys Ser PheLeu Lys Ala Gly Gly Glu 275 280 285 Val Ile Asp Ser Val Thr Trp His HisTyr Tyr Leu Asn Gly Arg Thr 290 295 300 Ala Thr Arg Glu Asp Phe Leu AsnPro Asp Val Leu Asp Ile Phe Ile 305 310 315 320 Ser Ser Val Gln Lys ValPhe Gln Val Val Glu Ser Thr Arg Pro Gly 325 330 335 Lys Lys Val Trp LeuGly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala 340 345 350 Pro Leu Leu SerAsp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys 355 360 365 Leu Gly LeuSer Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val 370 375 380 Phe PheGly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro 385 390 395 400Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr 405 410415 Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg 420425 430 Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly435 440 445 Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys TyrLeu 450 455 460 Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys TyrLeu Leu 465 470 475 480 Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys SerVal Gln Leu Asn 485 490 495 Gly Leu Thr Leu Lys Met Val Asp Asp Gln ThrLeu Pro Pro Leu Met 500 505 510 Glu Lys Pro Leu Arg Pro Gly Ser Ser LeuGly Leu Pro Ala Phe Ser 515 520 525 Tyr Ser Phe Phe Val Ile Arg Asn AlaLys Val Ala Ala Cys Ile 530 535 540 543 1721 nucleic acid Double linear3 CT AGA GCT TTC GAC 14 TCT CCG CTG CGC GGC AGC TGG CGG GGG GAG CAG CCAGGT GAG CCC AAG 62 ATG CTG CTG CGC TCG AAG CCT GCG CTG CCG CCG CCG CTGATG CTG CTG 110 Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu MetLeu Leu 5 10 15 CTC CTG GGG CCG CTG GGT CCC CTC TCC CCT GGC GCC CTG CCCCGA CCT 158 Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro ArgPro 20 25 30 GCG CAA GCA CAG GAC GTC GTG GAC CTG GAC TTC TTC ACC CAG GAGCCG 206 Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro35 40 45 CTG CAC CTG GTG AGC CCC TCG TTC CTG TCC GTC ACC ATT GAC GCC AAC254 Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn 5055 60 CTG GCC ACG GAC CCG CGG TTC CTC ATC CTC CTG GGT TCT CCA AAG CTT302 Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu 6570 75 80 CGT ACC TTG GCC AGA GGC TTG TCT CCT GCG TAC CTG AGG TTT GGT GGC350 Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly 8590 95 ACC AAG ACA GAC TTC CTA ATT TTC GAT CCC AAG AAG GAA TCA ACC TTT398 Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe 100105 110 GAA GAG AGA AGT TAC TGG CAA TCT CAA GTC AAC CAG GAT ATT TGC AAA446 Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys 115120 125 TAT GGA TCC ATC CCT CCT GAT GTG GAG GAG AAG TTA CGG TTG GAA TGG494 Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp 130135 140 CCC TAC CAG GAG CAA TTG CTA CTC CGA GAA CAC TAC CAG AAA AAG TTC542 Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe 145150 155 160 AAG AAC AGC ACC TAC TCA AGA AGC TCT GTA GAT GTG CTA TAC ACTTTT 590 Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe165 170 175 GCA AAC TGC TCA GGA CTG GAC TTG ATC TTT GGC CTA AAT GCG TTATTA 638 Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu180 185 190 AGA ACA GCA GAT TTG CAG TGG AAC AGT TCT AAT GCT CAG TTG CTCCTG 686 Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu195 200 205 GAC TAC TGC TCT TCC AAG GGG TAT AAC ATT TCT TGG GAA CTA GGCAAT 734 Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn210 215 220 GAA CCT AAC AGT TTC CTT AAG AAG GCT GAT ATT TTC ATC AAT GGGTCG 782 Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser225 230 235 240 CAG TTA GGA GAA GAT TAT ATT CAA TTG CAT AAA CTT CTA AGAAAG TCC 830 Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg LysSer 245 250 255 ACC TTC AAA AAT GCA AAA CTC TAT GGT CCT GAT GTT GGT CAGCCT CGA 878 Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln ProArg 260 265 270 AGA AAG ACG GCT AAG ATG CTG AAG AGC TTC CTG AAG GCT GGTGGA GAA 926 Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly GlyGlu 275 280 285 GTG ATT GAT TCA GTT ACA TGG CAT CAC TAC TAT TTG AAT GGACGG ACT 974 Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly ArgThr 290 295 300 GCT ACC AGG GAA GAT TTT CTA AAC CCT GAT GTA TTG GAC ATTTTT ATT 1022 Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile PheIle 305 310 315 320 TCA TCT GTG CAA AAA GTT TTC CAG GTG GTT GAG AGC ACCAGG CCT GGC 1070 Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr ArgPro Gly 325 330 335 AAG AAG GTC TGG TTA GGA GAA ACA AGC TCT GCA TAT GGAGGC GGA GCG 1118 Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly GlyGly Ala 340 345 350 CCC TTG CTA TCC GAC ACC TTT GCA GCT GGC TTT ATG TGGCTG GAT AAA 1166 Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp LeuAsp Lys 355 360 365 TTG GGC CTG TCA GCC CGA ATG GGA ATA GAA GTG GTG ATGAGG CAA GTA 1214 Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met ArgGln Val 370 375 380 TTC TTT GGA GCA GGA AAC TAC CAT TTA GTG GAT GAA AACTTC GAT CCT 1262 Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn PheAsp Pro 385 390 395 400 TTA CCT GAT TAT TGG CTA TCT CTT CTG TTC AAG AAATTG GTG GGC ACC 1310 Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys LeuVal Gly Thr 405 410 415 AAG GTG TTA ATG GCA AGC GTG CAA GGT TCA AAG AGAAGG AAG CTT CGA 1358 Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg ArgLys Leu Arg 420 425 430 GTA TAC CTT CAT TGC ACA AAC ACT GAC AAT CCA AGGTAT AAA GAA GGA 1406 Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg TyrLys Glu Gly 435 440 445 GAT TTA ACT CTG TAT GCC ATA AAC CTC CAT AAC GTCACC AAG TAC TTG 1454 Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val ThrLys Tyr Leu 450 455 460 CGG TTA CCC TAT CCT TTT TCT AAC AAG CAA GTG GATAAA TAC CTT CTA 1502 Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp LysTyr Leu Leu 465 470 475 480 AGA CCT TTG GGA CCT CAT GGA TTA CTT TCC AAATCT GTC CAA CTC AAT 1550 Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys SerVal Gln Leu Asn 485 490 495 GGT CTA ACT CTA AAG ATG GTG GAT GAT CAA ACCTTG CCA CCT TTA ATG 1598 Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr LeuPro Pro Leu Met 500 505 510 GAA AAA CCT CTC CGG CCA GGA AGT TCA CTG GGCTTG CCA GCT TTC TCA 1646 Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly LeuPro Ala Phe Ser 515 520 525 TAT AGT TTT TTT GTG ATA AGA AAT GCC AAA GTTGCT GCT TGC ATC TGA 1694 Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val AlaAla Cys Ile 530 535 540 543 AAA TAA AAT ATA CTA GTC CTG ACA CTG 1721 26nucleic acid single linear 4 CGCATATGCA GGACGTCGTG GACCTG 26 24 nucleicacid single linear 5 TATGATCCTC TAGTACTTCT CGAC 24 35 nucleic acidsingle linear 6 AGGAATTCAC CATGCTGCTG CGCTCGAAGC CTGCG 35 22 nucleicacid single linear 7 GAGTAGCAAT TGCTCCTGGT AG 22 35 nucleic acid singlelinear 8 GTCTCGAGAA AAGACAGGAC GTCGTGGACC TGGAC 35 59 nucleic acidsingle linear 9 AATTCACCAT GTCTGCACTT CTGATCCTAG CTCTTGTTGG AGCTGCAGTT50 GCTCAGGAC 59 53 nucleic acid single linear 10 CCTGAGCAAC TGCAGCTCCAACAAGAGCTA GGATCAGAAG TGCAGACTG GTG 53 52 nucleic acid single linear 11AATTCACCAT GTCTGCACTT CTGATCCTAG CTCTTGTTGG AGCTGCAGTT GC 52 50 nucleicacid single linear 12 CGGCAACTGC AGCTCCAACA AGAGCTAGGA TCAGAAGTGCAGACATGGTG 50 4 amino acid single linear 13 Ile Glu Gly Arg 4 5 aminoacid single linear 14 Asp Asp Asp Asp Lys 5 42 nucleic acid singlelinear 15 CCATCGATAG AAGGACGAAA AAAGTTCAAG AACAGCACCT AC 42 28 nucleicacid single linear 16 GGATCGATTG GTAGTGTTCT CGGAGTAG 28 39 nucleic acidsingle linear 17 GGATCGATAG AAGGACGATC TCAAGTCAAC CAGGATATT 39 32nucleic acid single linear 18 CCATCGATGC CCAGTAACTT CTCTCTTCAA AG 32 24nucleic acid single linear 19 TCAGATGCAA GCAGCAACTT TGGC 24 23 nucleicacid single linear 20 GAGCAGCCAG GTGAGCCCAA GAT 23 44 nucleic acidsingle linear 21 CCATCGATGA CGACGACAAG AAAAAGTTCA AGAACAGCAC CTAC 44 29nucleic acid single linear 22 GGATCGATCT GGTAGTGTTC TCGGAGTAG 29 43nucleic acid single linear 23 GGATCGATGA CGACGACAAG TCTCAAGTCAACCAGGATAT TTG 43 33 nucleic acid single linear 24 CCATCGATTT GGGAGTAACTTCTCTCTTCA AAG 33 5 amino acid single linear 25 Ser Gln Val Asn Gln 5

What is claimed is:
 1. A recombinantly made mammalian heparanase protein having an amino acid sequence at least 70% homologous to SEQ ID NO:2 and an apparent molecular weight as determined via denaturative gel electrophoresis of about 60-70 kDa wherein said recombinantly made heparanase can be activated by cleaving with a protease.
 2. A pharmaceutical composition comprising, as an active ingredients a recombinantly made mammalian heparanase protein having an amino acid sequence at least 70% homologous to SEQ ID NO:2 and an apparent molecular weight as determined via denaturative gel electrophoresis of about 60-70 kDa; and a pharmaceutically acceptable carrier wherein said recombinantly made heparanase can be activated by cleaving with a protease.
 3. A method of modulating heparin-binding of growth factors, cellular responses to heparin-binding growth factors and cytokines, cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and bacterial infections or disintegration of neurodegenerative plaques, the method comprising the step of administering to a subject a recombinantly made mammalian heparanase protein having an amino acid sequence at least 70% homologous to SEQ ID NO:2 and an apparent molecular weight as determined via denaturative gel electrophoresis of about 60-70 kDa wherein said recombinantly made heparanase can be activated by cleaving with a protease.
 4. A medical equipment comprising a medical device containing, as an active ingredient, a recombinantly made mammalian heparanase protein having an amino acid sequence at least 70% homologous to SEQ ID NO:2 and an apparent molecular weight as determined via denaturative gel electrophoresis of about 60-70 kDa wherein said recombinantly made heparanase can be activated by cleaving with a protease. 